CN103596983B - Anti-FGFR4 antibody and using method - Google Patents

Abstract

The present invention provides anti-FGFR4 antibodies and methods of use thereof.

Description

Anti-FGFR4 antibodies and methods of use

Cross References to Related Applications

This application claims priority to US Patent Application No. 61/473,106, filed April 7, 2011, the contents of which are hereby incorporated by reference.

sequence listing

This application, containing a Sequence Listing, has been filed via EFS-WEB in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 22, 2012, is named P4524R1WO.txt and is 37,020 bytes in size.

field of invention

The present invention relates to anti-FGFR4 antibodies and methods of use thereof.

Background of the invention

Fibroblast growth factors (FGFs) constitute a family of 22 structurally related polypeptides with distinct biological activities; most of these signaling molecules bind to and activate their cognate receptors (FGFR; designated FGFR1-4) (regulated by a family of somatic tyrosine kinases) to function (Eswarakumar et al., 2005; Ornitz and Itoh, 2001). These receptor-ligand interactions lead to receptor dimerization and autophosphorylation, complex formation with membrane-bound and cytoplasmic accessory proteins, and initiation of various signaling cascades (Powers et al., 2000). The FGFR-FGF signaling system plays an important role in development and tissue repair by regulating cellular functions/processes such as growth, differentiation, migration, morphogenesis, and angiogenesis.

Alterations (i.e., overexpression, mutations, translocations, and truncations) in FGFR have been associated with a variety of human cancers, including myeloma, breast, gastric, colon, bladder, pancreatic, and hepatocellular carcinomas (Bange et al .,2002; Cappellen et al.,1999; Chesi et al.,2001; Chesi et al.,1997; Gowardhan et al.,2005; Jaakkola et al.,1993; Jang et al.,2001; Jang et al. ,2000; Jeffers et al.,2002; Xiao et al.,1998). Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, causing more than 500,000 deaths per year (Shariff et al., 2009). Although the role of FGFR4 in cancer remains to be fully elucidated, several findings suggest that this receptor may be an important player in the development and/or progression of HCC. FGFR4 is the predominant FGFR isoform present in human hepatocytes (Kan et al., 1999); we have also previously reported that liver tissues have the highest transcript levels of FGFR4 (Lin et al., 2007). In addition to FGFR4, which is overexpressed in liver cancer (as well as several other types of human tumors), several missense genetic alterations were observed in HCC patient samples; notably, a highly frequent G388R mononucleotide in FGFR4 was identified Acid polymorphisms (associated with reduced survival in head and neck cancer, and a more aggressive phenotype in colon, soft tissue, prostate, and breast cancer) (Ho et al., 2009). Furthermore, it has been previously demonstrated that ectopic expression of FGF19, a specific ligand for FGFR4, in mice promotes hepatocyte proliferation, hepatocyte dysplasia, and neoplasia (Nichole et al., 2002).

It is clear that there remains a need for agents with clinical properties most suitable for development as therapeutic agents. The invention described herein fulfills this need and provides other benefits.

All references (including patent applications and publications) cited herein are incorporated by reference in their entirety.

Summary of the invention

The present invention provides anti-FGFR4 antibodies and methods of use thereof.

In one aspect, the invention provides an isolated antibody that binds FGFR4, wherein the anti-FGFR4 antibody binds human FGFR4 with an affinity < 1 nM. In some embodiments, the anti-FGFR4 antibody binds human, mouse, and macaque FGFR4 with an affinity < 1 nM. In some embodiments, the anti-FGFR4 antibody binds human FGFR4 with an affinity < 0.05 nM.

In some embodiments, the anti-FGFR4 antibody does not substantially bind to the mouse C3 protein having the amino acid sequence shown in Figure 12D.

In some embodiments, the anti-FGFR4 antibody binds denatured FGFR4. In some embodiments, the anti-FGFR4 antibody binds reduced, denatured FGFR4. In some embodiments, Western blotting is used to determine the binding of the anti-FGFR4 antibody to FGFR4.

In some embodiments, the anti-FGFR4 antibody does not substantially bind to human FGFR4 comprising a G165A mutation. In some embodiments, the anti-FGFR4 antibody binds to an antibody comprising, consisting essentially of, or consisting of amino acid numbers 150 to 170 of the mature human FGFR4 amino acid sequence, or consisting of amino acid numbers 150 to 170 of the mature human FGFR4 amino acid sequence. Polypeptides whose sequences consisting of 150 to 170 have at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity. In some embodiments, the anti-FGFR4 antibody binding comprises, consists essentially of, or consists of amino acid number 150 of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence to 170 composed peptides.

In some embodiments, the anti-FGFR4 antibody is an antagonist of FGFR4 activity. In some embodiments, the FGFR4 activity is FGF-induced cell proliferation, FGF binding to FGFR4, FGF19-mediated inhibition of CYP7α7 expression in cells exposed to FGF19, or FGF19-induced colony formation. In some embodiments, the binding of FGF1 and/or FGF19 to FGFR4 is inhibited. In some embodiments, inhibition of FGF1 binding to FGFR4 has an _IC50 of about 0.10 nM and inhibition of FGF19 binding of FGFR4 has an _IC50 of about 0.10 nM.

In some embodiments, the anti-FGFR4 antibody is a monoclonal antibody.

In some embodiments, the anti-FGFR4 antibody is a human antibody, a humanized antibody, or a chimeric antibody.

In some embodiments, the anti-FGFR4 antibody is an antibody fragment that binds FGFR4.

In some embodiments, the antibody comprises (a) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, (b) HVR-L3 comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-H2, It comprises the amino acid sequence of SEQ ID NO:2.

In some embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (c) HVR-H3, It comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the antibody further comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR- L3, which comprises the amino acid sequence of SEQ ID NO:6.

In some embodiments, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR-L3, It comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the antibody further comprises the light chain variable domain framework sequence of SEQ ID NO:9, 10, 11 and/or 12.

In some embodiments, the antibody further comprises the heavy chain variable domain framework sequence of SEQ ID NO: 13, 14, 15 and/or 16.

In some embodiments, the antibody comprises (a) a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:7; (b) a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:8 Sequence identity; or (c) the VH sequence in (a) and the VL sequence in (b). In some embodiments, the antibody comprises the VH sequence of SEQ ID NO:7. In some embodiments, the antibody comprises the VL sequence of SEQ ID NO:8.

In some embodiments, the antibody comprises a VH sequence of SEQ ID NO:7 and a VL sequence of SEQ ID NO:8.

In some embodiments, the antibody is a full length IgG1 antibody.

The invention also provides isolated nucleic acids encoding any of the antibodies described herein.

The invention also provides host cells comprising any of the nucleic acids described herein.

The invention also provides a method of producing an antibody comprising culturing a host cell described herein such that the antibody is produced. In some embodiments, the method further comprises recovering the antibody from the host cell.

The invention also provides immunoconjugates comprising any of the antibodies described herein and a cytotoxic agent.

The invention also provides a pharmaceutical formulation comprising any of the antibodies described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises additional therapeutic agents.

The invention also provides any antibody described herein for use as a medicament.

In some embodiments, the antibody is used to treat cancer.

In some embodiments, the antibodies are used to inhibit cell proliferation.

In some embodiments, the antibody is used in the preparation of a medicament.

In some embodiments, the medicament is used to treat cancer.

In some embodiments, the drug is used to inhibit cell proliferation.

The invention also provides a method of treating an individual having cancer comprising administering to the individual an effective amount of any of the antibodies described herein. In some embodiments, the method further comprises administering to the individual an additional therapeutic agent.

The invention also provides a method of inhibiting cell proliferation in an individual comprising administering to the individual an effective amount of any antibody described herein to inhibit cell proliferation.

In some embodiments, the cancer is breast cancer, lung cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, pituitary cancer, breast fibroadenoma, head and cervical cancer, soft tissue cancer, neuroblastoma, melanoma, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, and/or liver cancer.

In certain embodiments, the cancer exhibits FGFR4 expression (such as overexpression), amplification, or activation. In certain embodiments, the cancer exhibits FGFR4 amplification.

Brief description of the drawings

Figure 1: FGFR4 is required for FGF19-mediated liver tumorigenesis. A, Multiple, large, raised tumors (arrowheads) protruding from the liver surface of 10-month-old FGF19-TG:FGFR4-WT mice (left panel). Liver from a 10-month-old FGF19-TG:FGFR4-KO mouse (right panel). B, BrdU incorporation in female (left panel) and male (right panel) FGF19-TG or wild-type mice mated with FGFR4-KO or FGFR4-WT mice. C, Multiple, large, raised tumors (arrowheads) on the liver surface of 4-month-old DEN-treated FGF19-TG:FGFR4-WT mice. D, Prevalence of liver tumors in male and female FGF19-TG mice treated with DEN, as determined by macroscopic and histological examination. E, Liver weights from FGF19-TG or wild-type female (left panel) and male (right panel) mice treated with DEN. Asterisks (*) indicate that liver weights of DEN-treated male FGF19-TG mice could not be measured since the 7-month time point, as none survived beyond 6 months of age. F, Liver weight of FGF19-TG or wild-type female (left panel) and male (right panel) FGFR4-KO mice treated with DEN.

Figure 2: LD1 binds FGFR4. A, LD1 binds human (h), mouse (m), and macaque (c) FGFR4, but not hFGFR1, hFGFR2, or hFGFR3. Binding of LD1 to immobilized FGFR Fc chimeric protein was determined by a solid-phase binding assay. B, LD1 binding affinity to mouse, macaque, and human FGFR4, as determined by surface plasmon resonance. C, Binding of LD1 to hFGFR4 expressed on the cell surface of stably transfected HEK293 cells, determined by FACS (RFU=relative fluorescence units). D, Binding of LD1 to immobilized hFGFR4-Flag chimeric protein carrying a point mutation, measured by solid-phase binding assay. E, Binding of LD1 to hFGFR4-Flag chimeric protein carrying point mutations, assessed by Western blot. Mutant proteins were electrophoresed and immunoblotted sequentially using LD1, anti-FGFR4 (8G11), and anti-Flag antibodies. F, Dimer model showing the position of G165 (black) on FGFR4 (black and white) bound to FGF19 (grey).

Figure 3: LD1 inhibits FGFR4 activity. A, LD1 inhibits FGFR4 binding to FGF1 and FGF19, as determined by a solid-phase binding assay. B, LD1 inhibits FGF1-stimulated proliferation of BaF3 cells stably expressing FGFR4/R1. C, LD1 inhibits FGFR4 signaling in L6 cells stably expressing FGFR4. D, Cell surface expression of FGFR4 protein in a subset of liver tumor cell lines, as determined by FACS analysis using LD1.

Figure 4: LD1 inhibits FGFR4 biological activity in liver cancer cell lines. A, LD1 inhibits FGFR4 signaling in HEP3B cells, assessed by Western blot. B, LD1 inhibits FGFR4-regulated CYP7α1 expression in HEP3B cells. CYP7α1 levels are expressed as fold expression relative to the level in untreated cells. C, LD1 inhibits FGFR4-regulated c-Fos expression in a panel of HCC cell lines. Results are expressed as fold expression relative to c-Fos levels in untreated cells. D, Inhibition of FGFR4 expression inhibits colony formation in JHH5 cells stably transfected with FGFR4 shRNA doxycycline-inducible vector. E, LD1 inhibits colony formation in HCC cell lines. F, Counts of colony formation of LD1-inhibited hepatoma cell lines. Values are expressed as a percentage of the number of colonies counted without the addition of LD1.

Figure 5: In vivo efficacy of LD1. A, LD1 inhibits FGF19-regulated c-Fos expression in mouse liver. Results are expressed as fold expression relative to c-Fos levels in the liver of untreated mice. B, LD1 (30 mg/kg; twice weekly) inhibits HUH7 xenograft tumor growth in vivo. C, Effect of LD1 on mRNA expression of FGFR4, CYP7α1, c-Fos, and egr-1 in HUH7 xenograft tumors from Figure 5B. D, Multiple, large, raised tumors (arrows) protruding from the liver surface of DEN-accelerated FGF19-TG:FGFR4-WT mice treated with control antibody (upper panel). Liver of DEN-accelerated FGF19-TG:FGFR4-WT mice treated with LD1 (lower panels). E, Liver weight of DEN-accelerated FGF19-TG:FGFR4-WT mice treated with control antibody, LD1, or 1A6 (anti-FGF19 antibody).

Figure 6: FGFR4 expression is dysregulated in cancer. A, Whisker-box plot showing FGFR4 expression in human tumor and normal tissues, as determined by mRNA analysis of the BioExpress database. The center line indicates the median; the boxes represent the interquartile range between the first and third quartiles. The "lines" extend from the interquartiles to the extreme values. B, FGFR4 immunostaining in samples of breast (100X magnification) and pancreas (100X magnification) adenocarcinoma and hepatocellular carcinoma (200X magnification and 400X magnification). C, FGFR4 mRNA expression in a panel of human normal livers and liver tumors, as determined by qRT-PCR. Values for each sample are expressed as fold relative to the levels observed in sample N1.

Figure 7: FGFR expression in liver cancer cell lines. A, FGFR4 mRNA expression in a panel of liver tumor cell lines, as determined by qRT-PCR. Values are expressed as fold expression relative to FGFR1 levels in the JHH4 cell line. B, FGFR4 protein expression in the same panel of cell lines as in Figure 7A, as determined by Western blotting.

Figure 8: LD1 inhibits FGFR4 biological activity in HUH7 cells. LD1 inhibits FGFR4-regulated CYP7α1 repression in HUH7 cells. CYP7α1 levels are expressed as fold expression relative to the level in untreated cells.

Figure 9: In vivo efficacy of LD1. LD1 (30 mg/kg) inhibits HUH7 xenograft tumor growth in vivo. The antitumor efficacy of LD1 was assessed in a biweekly modality.

Figure 10: Variable domain sequences of mouse and humanized variants of anti-FGFR4. Amino acid sequences of mouse LD1 and humanized variants hLD1.vB and hLD1.v22 compared to (A) human kappa I (huKI) and (B) human VH subgroup III (huIII) used in trastuzumab Alignment of variable domain frames. Differences are highlighted with dashed boxes and positions are numbered according to Kabat. Based on a combination of sequence, structure, and contact CDR definitions (MacCallumRM et al., J of Molec Biol (1996); 262:732-45) selected from mouse LD1 grafted into human variable kappa I and subgroup III consensus frameworks Hypervariable regions are marked with boxes. Three vernier positions in the light chain were changed during humanization to restore affinity; these positions were not expected to be surface exposed.

Figure 11: Pharmacokinetics and distribution of anti-FGFR4 antibody variants. (A) Comparison of chLD1 and hLD1.vB binding to FGFR4 using FGFR4 ELISA. (B) Comparison of 16-day tumor volumes of chLD1, hLD1.vB and vehicle in the HUH7 human hepatocellular carcinoma xenograft model in CRL nu/nu mice. Antibodies were administered at 30 mg/kg twice a week (10 mice per group). Only chLD1 was effective in reducing tumor growth relative to PBS control (p-value=0.014), while hLD1.vB was not significantly effective (p-value=0.486). (C) Pharmacokinetics, chLD1 and hLD1.vBNCR nude mice were dosed IV with 1 or 20 mg/kg and samples were analyzed using FGFR4 ELISA. Similar results were obtained using IgG ELISA (not shown). (D) Tissue distribution ^of125I -chLD1 and125I- ^hLD1.vB in NCR nude mice. Mice were dosed with either ¹²⁵ I-chLD1 or ¹²⁵ I-hLD1.vB, and the percent injected dose per gram of tissue (%ID/g) was determined 2 hours after dosing, as described in Methods.

Figure 12: Identification of the interaction between hLD1.vB and mouse C3d. (A) Detection of chLD1 (hatched bars) and hLD1.vB (white bars) after incubation in PBS/BSA or NCR nude mouse, rat, human and macaque plasma for 48 hours. Percent recovery was determined using FGFR4 ELISA. (B) Plasma binding analysis ^of125I -chLD1 (solid line) ^and125I -hLD1.vB (dashed line). Traces are off-set; dots indicate the position of the 150 kDa peak. Antibodies were incubated in mouse plasma for 0 and 48 mice followed by analysis using size exclusion HPLC. See Figure 15 for incubations in PBS/BSA, human plasma or macaque plasma. All incubations produced a peak at the expected 150 kDa corresponding to IgG. Only higher molecular weight peaks were observed in mouse plasma samples containing hLD1.vB. Initial time points revealed additional peaks at approximately 270 kDa and approximately 550 kDa, while at 48 hours only the 270 kDa peak was observed. No high molecular weight peaks were observed upon incubation of hLD1.vB/mouse plasma at pH 4, indicating that the presence of these high molecular weight peaks is pH dependent (Figure 15). (C) Immunoprecipitation of mouse plasma. chLD1 and hLD1.vB were incubated in mouse plasma for 24 hours at 37°C and analyzed by size exclusion HPLC. Protein G beads were then added to the fractions, followed by SDS-PAGE analysis. A band at approximately 37 kDa was detected in the fraction corresponding to the 270 kDa peak present in hLD1.vB/mouse plasma samples (lane 3), but not in samples from macaque or human plasma or PBS/BSA (Figure 15 ). This band was not observed in mouse plasma alone (lane 4) or in mouse plasma incubated with chLD1 (lane 2). Lane 1 runs protein molecular weight markers. (D) MS/MS sequence coverage of mouse C3 obtained from hLD1.vB immunoprecipitation. The sequence of mouse C3 (SEQ ID NO:38) is shown and the region encoding C3d is underlined. The identified peptides are as follows:

DVPAADLSDQVPDTDSETRIILQGSPVVQMAEDAVDGER (SEQ ID NO: 17)

RQEALELIKKGYTQQLAFK (SEQ ID NO: 18)

AAFNNRPPSTWLTAYVVK (SEQ ID NO: 19)

AANLIAIDSHVLCGAVK (SEQ ID NO: 20)

QKPDGVFQEDGPVIHQEMIGGFR (SEQ ID NO: 21)

EADVSLTAFVLIALQEARDICEGQVNSLPGSINKAGEYIEASYMNLQRPYTVAIAGYALALMNK (SEQ ID NO: 22)

WEEPDQQLYNVEATSY (SEQ ID NO: 23)

YYGGGYGSTQATFMVFQALAQYQTDVPDHK (SEQ ID NO: 24)

GTLSVVAVYHAK (SEQ ID NO: 25)

DLELLASGVDR (SEQ ID NO: 26)

NTLIIYLEK (SEQ ID NO: 27).

Figure 13: Affinity matured anti-FGFR4 variants lack C3d binding. (A) Detection of FGFR4 binding, chLD1, hLD1.vB, and hLD1.v22 were incubated in NCR-null, C3 wild-type (wt) and C3ko mouse sera for 16 hours before assessment using FGFR4 ELISA. Samples were normalized to the same samples incubated in PBS/0.5%BSA. (B) C3d immunoprecipitation from hLD1.v22 deficient mice. Immunoprecipitations from NCR nude mouse plasma using chLD1 (lane 2), hLD1.vB (lane 3) and hLD1.v22 (lane 4) were analyzed by SDS-PAGE. The ~37 kDa band was only detected in hLD1.vB/mouse plasma samples. Lane 1 runs protein molecular weight markers.

Figure 14: Loss of C3d binding restores pharmacokinetics and efficacy. (A) Pharmacokinetic analysis of chLD1 and hLD1.vB in C3wt and C3ko mice. Antibodies were administered at 20 mg/kg IV; their concentrations in blood were monitored using FGFR4 ELISA. Clearance of hLD1.vB in C3ko mice was similar to clearance of chLD1 in both C3ko and C3wt mice. (B) Pharmacokinetic analysis of chLD1 hLD1.vB and hLD1.v22 in NCR nude mice. Antibodies were administered at 20 mg/kg IV; their concentrations in blood were monitored using FGFR4 ELISA. Clearance of hLD1.v22 was similar to that of chLD1. (C) Comparison of chLD1 hLD1.vB and hLD1.v22 in the HUH7 human HCC xenograft model in CRL nu/nu mice. Antibodies were dosed once a week at 30 mg/kg (10 mice per group) and tumor volumes were monitored for 4 weeks. On day 21, chLD1 and hLD1.v22 (p-value=7x10 ⁻⁷ and 3x10 ⁻⁵ , respectively) were effective in reducing tumor growth relative to PBS control (open squares), while hLD1.vB was only marginally effective (p-value=0.011).

Figure 15: Size exclusion HPLC analysis of radiolabeled chLD1 and hLD1.vB in plasma of samples from in vitro (AB) and in vivo studies (C). ¹²⁵ I-hLD1.vB (A) and ¹²⁵ I-chLD1 (B) were added to PBS/BSA or mouse, human and macaque plasma and analyzed by size exclusion HPLC at 0 or 48 hours. All traces reveal an IgG peak at expected 150 kDa (right peak). Higher molecular weight peaks at approximately 270 kDa (middle peak) and approximately 550 kDa (left peak) were observed only in the initial mouse plasma sample containing hLD1.vB; at 48 hours, only an additional 270 kDa peak was observed. These high molecular weight peaks were not observed for hLD1.vB in mouse plasma at pH 4.0, indicating that the generation of high molecular weight peaks is pH dependent. No high molecular weight peaks were detected in macaque or human plasma or in any chLD1-supplemented plasma; (C) In vivo mouse plasma samples. The specific activity of chLD1 was 12.52 μCi/μg and hLD1.vB was 9.99 μCi/ug after administration of ~0.1 mg/kg antibody to mice. Serum samples were collected at 0.25, 2, 5, 24, 72 and 120 hours. All traces reveal a peak at the expected 150 kDa for peak IgG (right peak). Higher molecular weight peaks at -270 kDa (middle peak) and -550 kDa (left peak) were observed only in hLD1.vB serum samples. No high molecular weight peak was observed for chLD1.

Figure 16: Immunoprecipitation and SDS-PAGE analysis. Antibodies were incubated in plasma at 37°C for 24 hours and then fractionated by size exclusion HPLC. Protein G beads were added to the size exclusion HPLC fraction containing the high molecular weight peak. Pull-down samples were analyzed by SDS-PAGE. (A) In vitro rat plasma; (B) In vitro macaque plasma; (C) In vitro human plasma. A -37 kDa protein was detected in rat samples incubated with hLD1.vB, but not in macaque and human samples (lane 4 of their respective gels). No band for the ~37 kDa protein was observed in any of the blank plasma (lane 2 of each gel) or in the chLD1 samples (lane 3 of each gel). Lane 1 runs protein molecular weight markers. (D) In vivo mouse plasma. Mice were dosed with hLD1.vB or chLD1 as described in Methods. Protein G beads were then added to serum samples collected 2 hours from the dosing time point and analyzed by SDS-PAGE. A band at ~37 kDa was observed in hLD1.vB (lane 3), but not in chLD1 serum samples (lane 2). Lane 1 runs protein molecular weight markers.

Figure 17: ELISA detection of chLD1 and hLD1.vB added to C3wt serum, C3ko mouse serum, and PBS/BSA. Antibodies were incubated overnight in serum or PBS/BSA before analysis by ELISA. Relative to PBS/BSA, hLD1.vB was completely recovered in C3ko mouse sera, but not in C3wt mouse sera. chLD1 was fully recovered from all matrices.

Figure 18: Shows an exemplary human FGFR4 amino acid sequence of GenBank accession number AAB25788.

Detailed description of the invention

I. Definition

For the purposes herein, an "acceptor human framework" refers to a framework comprising a variable light chain (VL) domain or a variable heavy domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework as defined below. frame of the amino acid sequence. An acceptor human framework "derived" from a human immunoglobulin framework or a human consensus framework may comprise its identical amino acid sequence, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less , or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

"Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) that result in the antibody being more sensitive to the antigen as compared to a parent antibody that does not possess such alterations. Affinity improved.

Anti-angiogenic agents refer to compounds that block or to some extent interfere with the development of blood vessels. Anti-angiogenic factors can be, for example, small molecules or antibodies that bind growth factors or growth factor receptors involved in promoting angiogenesis. In one embodiment, the anti-angiogenic agent is an antibody that binds vascular endothelial growth factor (VEGF), such as bevacizumab

The terms "anti-FGFR4 antibody" and "antibody that binds FGFR4" refer to an antibody that is capable of binding FGFR4 with sufficient affinity such that the antibody can be used to target FGFR4 as a diagnostic and/or therapeutic agent. In one embodiment, the extent to which an anti-FGFR4 antibody binds an unrelated, non-FGFR4 protein is less than about 10% of the binding of the antibody to FGFR4, as measured, eg, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds FGFR4 has ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, such as 10 ⁻⁸ M to 10 ^-13 M, such as 10 ^-9 M to 10 ^-13 M) dissociation constant (Kd). In certain embodiments, an anti-FGFR4 antibody binds an epitope of FGFR4 that is conserved among FGFR4 from different species.

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, so long as they exhibit expected antigen-binding activity.

"Antibody fragment" refers to a molecule that is distinct from an intact antibody, comprises a portion of an intact antibody and binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); Sexual antibodies.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen in a competition assay. Antigen binding is blocked by 50% or more. Exemplary competition assays are provided herein.

As used herein, the term "FGFR4" refers to any native FGFR4 from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats), unless otherwise stated. The term encompasses "full length", unprocessed FGFR4 as well as any form of FGFR4 that results from processing in the cell. The term also encompasses naturally occurring variants of FGFR4, such as splice variants or allelic variants. The amino acid sequence of an exemplary human FGFR4 is shown in FIG. 18 .

"FGFR4 activation" refers to the activation or phosphorylation of the FGFR4 receptor. In general, FGFR4 activation results in signal transduction (eg by the intracellular kinase domain of the FGFR4 receptor, phosphorylation of tyrosine residues in FGFR4 or substrate polypeptides). FGFR4 activation can be mediated by binding of the FGFR4 ligand (Gas6) to the FGFR4 receptor of interest. Binding of Gas6 to FGFR4 can activate the kinase domain of FGFR4 and thereby lead to phosphorylation of tyrosine residues in FGFR4 and/or phosphorylation of tyrosine residues in other substrate polypeptides.

The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (eg, Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, Cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer , thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

The terms "cell proliferative disorder" and "proliferative disorder" refer to a disorder associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

"Chemotherapeutic agent" refers to a chemical compound that is useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide Alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodepa ), carboquone, meturedepa, and uredepa; ethyleneimines and methylmelamines, including altretamine , triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenin (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, ); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog topotecan ) CPT-11 (irinotecan, ), acetylcamptothecin, scopoletin (scopoletin) and 9-aminocamptothecin); bryostatin (bryostatin); callystatin; CC-1065 (including its adozelesin ( carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; 1 and cryptophyllin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; spongistatin); nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide , mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine ), trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine , lomustine (lomustine), nimustine (nimustine) and ramustine (ranimustine); antibiotics such as enediyne antibiotics (enediyne) (such as calicheamicin (calicheamicin), especially Calicheamicin γ1I and calicheamicin ωI1 (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral α-4 integrin protein inhibitors; anthracyclines (dynemicins), including dynemicin A; esperamicins; and neocarzinostats in) chromophores and related chromoproteins (enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, Bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, Daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine, doxorubicin (including Morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolidodoxorubicin, doxorubicin hydrochloride liposome injection Liposome Doxorubicin TLC D-99 PEGylated liposomal doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as Mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin ), triiron doxorubicin (quelamycin), rhodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine tegafur Capecitabine Epothilone and 5-fluorouracil (5-FU); folate analogs such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitabine Azacitidine, 6-azuridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine , floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone ); anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone ; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; Lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; Toxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitracrin e); pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine ; Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid ; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verrucarin A, rod roridin A and anguidin); urethan; vindesine ( ); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ) (“Ara-C”); thiotepa; taxoids such as paclitaxel Albumin-modified nanoparticle formulations of paclitaxel (ABRAXANE ^TM ) and docetaxel (doxetaxel) Chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (For example ) and carboplatin; vincas, which prevent the polymerization of tubulin to form microtubules, including vinblastine vincristine Vindesine and vinorelbine Etoposide (VP-16); Ifosfamide; Mitoxantrone; Leucovorin; Novantrone; Edatrexate; Daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, Such as retinoic acid, including bexarotene Bisphosphonates (bisphosphonates), such as clodronate (clodronate) (eg or ), etidronate NE-58095, zoledronic acid/zoledronate alendronate Pamidronate tiludronate or risedronate and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation Nucleotides, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as Vaccines and gene therapy vaccines, such as vaccine, vaccines and Vaccines; topoisomerase 1 inhibitors (eg ); rmRH (eg ); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, Pfizer); perifosine, COX-2 inhibitors (eg, celecoxib or etoricoxib), proteosome inhibitors (eg, PS341); bortezomib CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersensodium pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, ); farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP ( Cyclophosphamide, Doxorubicin, Vincristine, and Prednisolone Combination Therapy) and FOLFOX (abbreviation for Oxaliplatin (ELOXATIN ^TM ) in combination with 5-FU and Leucovorin).

Chemotherapeutic agents as defined herein include the class of "antihormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that can promote cancer growth. They can be hormones themselves, including but not limited to: antiestrogens with mixed agonist/antagonist properties including tamoxifen (NOLVADEX), 4-hydroxytamoxifen, toremifene (toremifene) idoxifene, droloxifene, raloxifene Trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs), such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant (fulvestrant) and EM800 (agents that block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, Such as formestane and exemestane and nonsteroidal aromatase inhibitors, such as anastrozole Letrozole and aminoglutethimide, and other aromatase inhibitors, including vorozole megestrol acetate Fadrozole and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists, including leuprolide ( and ), goserelin, buserelin, and triptorelin; sex steroids, including progestines, such as megestrol acetate and acetic acid medroxyprogesteroneacetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all trans Retinoic acid and fenretinide; onapristone; antiprogestins; estrogen receptor down-regulators (ERDs); antiandrogens such as flutamide (flutamide), nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains is derived from a particular source or species, while the remaining portion of the heavy and/or light chains is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ , and IgA ₂ . The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

The term "cytostatic" refers to a compound or composition that arrests the growth of cells, either in vitro or in vivo. Thus, a cytostatic agent can be an agent that significantly reduces the percentage of cells in S phase. Other examples of cytostatic agents include agents that block cell cycle progression by inducing G0/G1 arrest or M phase arrest. Humanized anti-Her2 antibody trastuzumab is an example of a cytostatic agent that induces G0/G1 arrest. Classic M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin (doxorubicin), epirubicin, daunorubicin, etoposide, and bleomycin. Certain G1-blocking agents also spill over into S-phase arrest, such as DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, dichloroethylmethylamine ( mechlorethamine), cisplatin, methotrexate, 5-fluorouracil, and ara-C. For more information see Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cellcycle regulation, oncogenes, and antieioplastic drugs", Murakaini et al., WBSaunders, Philadelphia, 1995, e.g., p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs derived from the yew tree. Docetaxel derived from European yew ( Rhone-Poulenc Rorer) is Palitase ( A semisynthetic analogue of Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of mitosis in cells.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioactive isotopes (such as those of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu); chemotherapeutic agents or drugs (such as methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents); growth inhibitors; Enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agent.

A cancer or biological sample that "displays FGFR4 expression, amplification, or activation" refers to a cancer or biological sample that expresses (including overexpresses) FGFR4 in a diagnostic test, has an amplified FGFR4 gene, and/or otherwise exhibits FGFR4 activation or phosphorylation cancer or biological samples.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody that vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; body) downregulation; and B cell activation.

An "effective amount" of a medicament (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. Typically, the FRs of a variable domain consist of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms "full-length antibody", "intact antibody", and "whole antibody" are used interchangeably herein to refer to a heavy chain having a structure substantially similar to that of a native antibody or having an Fc region as defined herein antibodies.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A "human antibody" refers to an antibody that possesses an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using the human antibody repertoire or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

"Human consensus framework" refers to a framework representing the most frequently occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, a subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vol. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, usually two, substantially all variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all All FRs above correspond to those of human antibodies. Optionally, a humanized antibody can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, eg, a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a native 4-chain antibody contains six HVRs; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter of which are of the highest sequence variability and/or are involved in antigen recognition. Exemplary hypervariable regions exist at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 ( H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) are present at amino acid residues 24-34 of L1, 50-56 of L2, 89- 97. 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991)). With the exception of CDR1 in VH, the CDRs generally contain amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are the residues that contact the antigen. The SDR is contained within a CDR region known as the shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues of L1 Bases 31-34, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)). HVR residues and other residues (eg, FR residues) in variable domains are numbered herein according to Kabat et al., supra, unless otherwise indicated.

"Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

"Inhibiting cell growth or proliferation" means reducing the growth or proliferation of cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% , but also induces cell death.

"Isolated antibody" refers to an antibody that has been separated from components of its natural environment. In some embodiments, antibodies are purified to greater than 95% or 99% purity, such as by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase determined by HPLC). For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B848:79-87 (2007).

"Isolated nucleic acid" refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-FGFR4 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or in different vectors, and present in a host cell One or more positions of such nucleic acid molecules.

As used herein, the phrase "little to no binding" with respect to an antibody of the invention means that the antibody does not exhibit binding in a biologically meaningful amount. As will be understood in the art, the amount of activity can be determined quantitatively or qualitatively, so long as a comparison between an antibody of the invention and a reference counterpart can be made. Activity can be measured or detected according to any assay or technique known in the art, such as those described herein. For antibodies of the invention and their reference counterparts, the amount of activity can be determined in parallel or in separate runs.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, containing naturally occurring mutations or Apart from possible variant antibodies that occur during the production of monoclonal antibody preparations, such variants are generally present in very small amounts. Unlike polyclonal antibody preparations, which generally contain different antibodies directed against different determinants (epitopes), monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen. As such, the modifier "monoclonal" indicates the properties of an antibody acquired from a population of substantially homogeneous antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the invention can be produced by a variety of techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals containing all or part of the human immunoglobulin loci Such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radioactive label. Naked antibodies may be present in pharmaceutical formulations.

"Native antibody" refers to naturally occurring immunoglobulin molecules of varying structure. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains disulfide-bonded. From N to C-terminus, each heavy chain has a variable region (VH), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also called variable light domain or light chain variable domain, followed by a constant light (CL) domain. Depending on the amino acid sequence of their constant domains, antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ).

The term "package insert" is used to refer to the instructions commonly included in commercial packages of therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapies, contraindications and/or warnings concerning the use of such therapeutic products .

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity, and when any conservative substitutions are not considered part of the sequence identity, The percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence. Alignment for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. However, for the purposes of the present invention, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, along with user documentation, has been filed with the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source. The ALIGN2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

In the case of comparing amino acid sequences using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A relative to (to), with (with), or against (against) a given amino acid sequence B (or can be expressed as A given amino acid sequence A) having or comprising a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows:

Fraction X/Y times 100

where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with respect to B will not be equal to the % amino acid sequence identity of B with respect to A. Unless expressly stated otherwise, all % amino acid sequence identity values used herein were obtained using the ALIGN-2 computer program as described in the preceding paragraph.

The term "pharmaceutical formulation" refers to a form that allows the biological activity of the active ingredient contained therein to be effective and free of additional components that would be unacceptably toxic to a subject to whom the formulation would be administered preparations.

"Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

As used herein, "treatment" (and its grammatical variants such as "treatment" or "disposition") refers to clinical intervention that attempts to alter the natural course of the individual being treated, either for prophylaxis or during the course of clinical pathology conduct. Desired effects of treatment include, but are not limited to, prevention of occurrence or recurrence of disease, alleviation of symptoms, attenuation of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing of the rate of disease progression, amelioration or palliation of disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset/progression of a disease, or to slow the progression of a disease.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive when referred to herein.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in the binding of the antibody to antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, where each domain contains 4 conserved framework regions (FR) and 3 hypervariable regions (HVR). (See eg Kindt et al. Kuby Immunology, 6th Ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind a particular antigen can be isolated by screening a library of complementary VL or VH domains using the VH or VL domains, respectively, from the antibody that binds the antigen. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

A "VH subgroup III consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable heavy subgroup III of Kabat et al. In one embodiment, the VH subgroup III consensus framework amino acid sequence comprises at least part or all of each of the following sequences: EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 13)-H1-WVRQAPGKGLEWV (SEQ ID NO: 14)-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 15)-H3-WGQGTLVTVSS (SEQ ID NO: 16).

The "VL subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences in the variable light kappa subgroup I of Kabat et al. In one embodiment, the VL subgroup I consensus framework amino acid sequence comprises at least part or all of each of the following sequences: DIQMTQSPSSLSASSVGDRVTITC (SEQ ID NO:28)-L1-WYQQKPGKAPKLLIY (SEQ ID NO:29)-L2-GVPSRFSGSGSGTDFLTISSLQPEDFATYYC( SEQ ID NO:30)-L3-FGQGTKVEIK (SEQ ID NO:31).

The term "wasting/depleting" disorder (eg wasting syndrome, cachexia, sarcopenia) refers to a disorder caused by unwanted and/or unhealthy weight loss or loss of somatic cell mass. In the elderly, as well as in AIDS and cancer patients, wasting disease can lead to unwanted weight loss, including both fat and fat-free compartments. Wasting disease can result from improper diet and/or metabolic changes associated with malaise and/or the aging process. Cancer patients and AIDS patients, as well as those who have undergone extensive surgery or have chronic infection, immunological disease, hyperthyroidism, Crohn's disease, psychological disease, chronic heart failure, or other severe trauma, often suffer from wasting disease and sometimes Known as cachexia, metabolic disorders, and eating disorders. Cachexia is also manifested by hypermetabolism and hypercatabolism. Although cachexia and wasting disease are often used interchangeably to refer to a wasting disorder, at least one study distinguishes cachexia from wasting syndrome by the loss of fat-free mass, particularly somatic mass (Mayer, 1999, J. Nutr. 129 (1S Suppl.):256S-259S). Sarcopenia, another such condition affecting aging individuals, is often manifested by loss of muscle mass. As noted above, terminal wasting disease can occur in individuals with cachexia or sarcopenia.

II. Compositions and Methods

In one aspect, the invention is based in part on the identification of various FGFR4 binding agents, such as antibodies and fragments thereof. In certain embodiments, antibodies that bind FGFR4 are provided. Antibodies of the present invention are useful, for example, in the diagnosis and treatment of cancer, liver disease, and wasting (wasting disease).

A. Exemplary Anti-FGFR4 Antibodies

In one aspect, the invention provides isolated antibodies that bind FGFR4.

In certain embodiments, the anti-FGFR4 antibody binds human FGFR4 with an affinity < 1 nM. In some embodiments, the anti-FGFR4 antibody binds human FGFR4 with an affinity < 0.05 nM.

In certain embodiments, the anti-FGFR4 antibody binds mouse FGFR4 with an affinity < 1 nM.

In certain embodiments, the anti-FGFR4 antibody binds macaque FGFR4 with an affinity < 1 nM.

In certain embodiments, the anti-FGFR4 antibody binds human, mouse and macaque FGFR4 with an affinity < 1 nM.

In certain embodiments, the anti-FGFR4 antibody does not substantially bind human FGFR1, human FGFR2, and/or human FGFR3. In a certain embodiment, the anti-FGFR4 antibody exhibits little or no binding to human FGFR1, human FGFR2 and/or human FGFR3.

In certain embodiments, the anti-FGFR4 antibody does not substantially bind to a mouse C3 protein (in some embodiments, a mouse C3 protein having the amino acid sequence shown in Figure 12D). In a certain embodiment, the anti-FGFR4 antibody exhibits little or no binding to a mouse C3 protein (in some embodiments, a mouse C3 protein having the amino acid sequence shown in Figure 12D).

In certain embodiments, the anti-FGFR4 antibody is a humanized anti-FGFR4 antibody, wherein the antibody has a monovalent affinity for human FGFR4 comparable to that of a murine antibody comprising the light chain and heavy chain variable sequences shown in SEQ ID NOs: 8 and 7, respectively. The monovalent affinities of the antibodies are essentially the same. In some embodiments, the anti-FGFR4 antibody is a humanized and affinity matured antibody.

In certain embodiments, the anti-FGFR4 antibody is an antagonist of FGFR4 activity.

In certain embodiments, the anti-FGFR4 antibody inhibits FGF binding to FGFR4. In some embodiments, the binding of FGF1 and/or FGF19 to FGFR4 is inhibited. In some embodiments, the inhibition of FGF1 binding to FGFR4 has an _IC50 of about 0.10 nM. In some embodiments, the inhibition of FGF19 binding to FGFR4 has an _IC50 of about 0.10 nM.

In certain embodiments, an anti-FGFR4 antibody inhibits cell proliferation. In some embodiments, the proliferation is FGF-induced cell proliferation. In some embodiments, the cell proliferation is BAF3/FGFR4 transgenic cell proliferation.

In certain embodiments, the anti-FGFR4 antibody inhibits FGF (eg, FGF1 ) stimulated proliferation of FGFR4-expressing cells. In some embodiments, the cells are HUH7 cells.

In certain embodiments, the anti-FGFR4 antibody inhibits FGF19-mediated inhibition of CYP7α7 expression in cells exposed to FGF19.

In certain embodiments, the anti-FGFR4 antibody inhibits FGF19-induced phosphorylation of FGFR4, MAPK, FRS2, and/or ERK2 in cells exposed to FGF19.

In certain embodiments, the anti-FGFR4 antibody inhibits FGF19-induced colony formation. In some embodiments, the colony formation is HCC cell line colony formation. In some embodiments, the HCC cell line is JHH5.

In certain embodiments, the anti-FGFR4 antibody binds denatured FGFR4. In some embodiments, the anti-FGFR4 antibody binds reduced, denatured FGFR4. In some embodiments, binding to reduced, denatured FGFR4 is determined using Western blotting.

In certain embodiments, the anti-FGFR4 antibody binds to FGFR4 expressed on the surface of a cell. In some embodiments, the cells are HUH7 or JHH5 cells.

In certain embodiments, the anti-FGFR4 antibody does not substantially bind to human FGFR4 comprising a G165A mutation. In certain embodiments, the anti-FGFR4 antibody exhibits little or no binding to human FGFR4 comprising a G165A mutation.

In certain embodiments, the anti-FGFR4 antibody binding comprises, consists essentially of, or consists of amino acid number 150 of amino acid number 150 to 170 of the mature human FGFR4 amino acid sequence to 170 composed peptides. In certain embodiments, the anti-FGFR4 antibody binding comprises, consists essentially of, or consists of amino acid number 145 of amino acid number 145 to 180 of the mature human FGFR4 amino acid sequence. to 180 composed peptides.

In certain embodiments, the anti-FGFR4 antibody binds to an antibody comprising, consisting essentially of, or consisting of amino acid numbers 150 to 170 of the mature human FGFR4 amino acid sequence, or consisting of amino acid numbers 150 to 170 of the mature human FGFR4 amino acid sequence. Polypeptides whose sequences consisting of 150 to 170 have at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity. In certain embodiments, the anti-FGFR4 antibody binds to an antibody comprising, consisting essentially of, or consisting of amino acid numbers 145-180 of the mature human FGFR4 amino acid sequence, or consisting of amino acid numbers 145-180 of the mature human FGFR4 amino acid sequence. A polypeptide comprising a sequence consisting of 145 to 180 having at least 70%, 80%, 90%, 95%, 98% sequence identity or similarity.

In certain embodiments, an anti-FGFR4 antibody inhibits FGFR4 dimerization.

In certain embodiments, the anti-FGFR4 antibody binds at the FGFR4 dimerization interface.

In certain embodiments, an anti-FGFR4 antibody inhibits tumor growth. In some embodiments, the tumor growth is a liver tumor growth.

In one aspect, the invention provides an anti-FGFR4 antibody comprising at least one, two, three, four, five, or six HVRs selected from: (a) HVR-H1 comprising the amino acid sequence NHWMN (SEQ ID NO: 1); (b) HVR-H2, which comprises the amino acid sequence MILPVDSETTLEQKFKD (SEQ ID NO: 2); (c) HVR-H3, which comprises the amino acid sequence GDISLFDY (SEQ ID NO: 3); ( d) HVR-L1 comprising the amino acid sequence RTSQDISNFLN (SEQ ID NO:4); (e) HVR-L2 comprising the amino acid sequence YTSRLHS (SEQ ID NO:5); and (f) HVR-L3 comprising the amino acid Sequence QQGNALPYT (SEQ ID NO: 6).

In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH HVR sequences selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:2; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:3. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, and HVR-L3 comprising the amino acid sequence of SEQ ID NO:6. In yet another embodiment, the antibody comprises HVR-H3, HVR-L3 and HVR-L2, the HVR-H3 comprises the amino acid sequence of SEQ ID NO:3, the HVR-L3 comprises the amino acid sequence of SEQ ID NO:6, the HVR -H2 comprises the amino acid sequence of SEQ ID NO:2. In yet another embodiment, the antibody comprises: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2; and (c) HVR - H3 comprising the amino acid sequence of SEQ ID NO:3.

In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL HVR sequences selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4 (b) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:5; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:6. In one embodiment, the antibody comprises: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:4; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:5; and (c) HVR- L3, which comprises the amino acid sequence of SEQ ID NO:6.

In another aspect, an antibody of the invention comprises: (a) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from: (i) HVR-H1 comprising amino acids the sequence of SEQ ID NO: 1, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and (b) the VL domain, It comprises at least one, at least two, or all three VL HVR sequences selected from: (i) HVR-L1, which comprises the amino acid sequence SEQ ID NO: 4, (ii) HVR-L2, which comprises the amino acid The sequence of SEQ ID NO:5, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:6.

In another aspect, the present invention provides an antibody comprising: (a) HVR-H1, which comprises the amino acid sequence of SEQ ID NO: 1; (b) HVR-H2, which comprises the amino acid sequence of SEQ ID NO: 2; ( c) HVR-H3, which comprises the amino acid sequence of SEQ ID NO:3; (d) HVR-L1, which comprises the amino acid sequence of SEQ ID NO:4; (e) HVR-L2, which comprises the amino acid sequence of SEQ ID NO:5 sequence; and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:6.

In another aspect, the anti-FGFR4 antibody comprises an amino acid sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTNHWMNWVRQAPGKGLEWVGMILPVDSETTLEQKFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCTRGDISLFDYWGQGTLVTVSS (SEQ ID NO: 7) that is at least 90%, 91%, 92%, 93%, 94%, 9%, 9%, 95% identical , or heavy chain variable domain (VH) sequences with 100% sequence identity. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution relative to a reference sequence (eg conservative substitution), insertion, or deletion, but the anti-FGFR4 antibody comprising this sequence retains the ability to bind to FGFR4. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO:7. In certain embodiments, substitutions, insertions, or deletions occur in regions other than HVRs (ie, in FRs). Optionally, the anti-FGFR4 antibody comprises the VH sequence in SEQ ID NO: 7, including post-translational modifications of this sequence. In a particular embodiment, the VH comprises one, two or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1, (b) HVR-H2, It comprises the amino acid sequence of SEQ ID NO:2, and (c) HVR-H3 comprises the amino acid sequence of SEQ ID NO:3.

In another aspect, an anti-FGFR4 antibody is provided, wherein the antibody comprises at least 90%, 91%, 92%, 93%, 94%, 995%, 96%, %, 98%, 99%, or 100% sequence identity of the light chain variable domain (VL). In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity comprises a substitution relative to a reference sequence (eg conservative substitution), insertion, or deletion, but the anti-FGFR4 antibody comprising this sequence retains the ability to bind to FGFR4. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO:8. In certain embodiments, substitutions, insertions, or deletions occur in regions other than HVRs (ie, in FRs). Optionally, the anti-FGFR4 antibody comprises the VL sequence in SEQ ID NO: 8, including post-translational modifications of this sequence. In a particular embodiment, the VL comprises one, two or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4; (b) HVR-L2, It comprises the amino acid sequence of SEQ ID NO:5; and (c) HVR-L3, which comprises the amino acid sequence of SEQ ID NO:6.

In another aspect, an anti-FGFR4 antibody is provided, wherein the antibody comprises the VH of any of the embodiments provided above and the VL of any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO:7 and SEQ ID NO:8, respectively, including post-translational modifications of those sequences.

In any of the above embodiments, the anti-FGFR4 antibody is humanized. In one embodiment, an anti-FGFR4 antibody comprises the HVR of any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. Antibodies of the invention may comprise any suitable framework variable domain sequences provided that binding activity to FGFR4 is substantially retained, for example, in some embodiments, antibodies of the invention comprise human subgroup III heavy chain framework consensus sequences. In one embodiment of these antibodies, the framework consensus sequence comprises substitutions at position 71, 73, and/or 78. In some embodiments of these antibodies, position 71 is A, position 73 is T and/or position 78 is A. In one embodiment, these antibodies comprise huMAb4D5-8 ( Genentech, Inc., South San Francisco, CA, USA) (see also U.S. Patent Nos. 6,407,213 and 5,821,337 and Lee et al., J. Mol. Biol. (2004) 340(5): 1073-1093) Heavy chain variable domain framework sequence. In one embodiment, the framework sequence comprises the following acceptor human framework: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:9)-L1-WYQQKPGKAFKILIS (SEQ ID NO:10)-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:11)-L3-FGQGTKVEIK( SEQ ID NO: 12).

In yet another aspect, the invention provides anti-FGFR4 antibodies that bind to the same epitope as the anti-FGFR4 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as an anti-FGFR4 antibody comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO: 8 are provided. In certain embodiments, antibodies that bind to an epitope within the fragment of FGFR4 consisting of amino acids 145-180 of the sequence shown in Figure 18 (SEQ ID NO:39) are provided.

In yet another aspect, the present invention provides an anti-FGFR4 antibody that competes for binding to human FGFR4 with an anti-FGFR4 antibody comprising a VH sequence of SEQ ID NO:7 and a VL sequence of SEQ ID NO:8.

In yet another aspect of the invention, the anti-FGFR4 antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-FGFR4 antibody is an antibody fragment, such as a Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full length antibody, such as a whole IgGl antibody or other antibody class or isotype, as defined herein.

In yet another aspect, an antibody according to any of the above embodiments may incorporate, singly or in combination, any of the features described in sections 1-7 below:

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 ⁻⁸ M or less, such as 10 ⁻⁸ M to 10 ⁻¹³ M, eg, 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab format of the antibody of interest and its antigen as described in the assay described below. The solution-binding affinity of the Fab for antigen was measured by equilibrating the Fab with a minimal concentration of ( ^125I ) labeled antigen in the presence of a titration series of unlabeled antigen, and then capturing the bound antigen with an anti-Fab antibody-coated plate (see e.g. Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the conditions for the assay, the Multiwell plates (Thermo Scientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by 2% (w/v) bovine serum albumin in PBS at Block at room temperature (about 23°C) for 2-5 hours. In non-adsorbent plates (Nunc #269620), mix 100 pM or 26 pM 125I-antigen with serially diluted Fab of interest (for example with Presta et al., Cancer Res. 57:4593-4599 (1997) anti-VEGF antibody, Fab-12 assessment agree) mixed. The Fab of interest is then incubated overnight; however, the incubation can be continued for a longer period of time (eg, about 65 hours) to ensure equilibrium is reached. Thereafter, the mixture is transferred to a capture plate and incubated at room temperature (eg, 1 hour). The solution was then removed and replaced with 0.1% polysorbate 20 in PBS Wash the plate 8 times. After the plates had dried, 150 μl/well scintillation fluid (MICROSCINT-20 ^™ ; Packard) was added and the plates were counted for 10 minutes on a TOPCOUNT ^™ gamma counter (Packard). Concentrations of each Fab giving less than or equal to 20% of maximal binding were selected for use in competitive binding assays.

According to another embodiment, Kd is determined using surface plasmon resonance -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) measured at about 10 response units (RU) at 25°C using an immobilized antigen CM5 chip. Briefly, the carboxylate was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Methylated dextran biosensor chip (CM5, BIACORE, Inc.). Antigen was diluted to 5 μg/ml (approximately 0.2 μM) with 10 mM sodium acetate pH 4.8 and then injected at a flow rate of 5 μl/min to obtain approximately 10 response units (RU) of coupled protein. After antigen injection, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, two- ^fold serial dilutions of Fab (0.78 nM to 500nM). Using a simple one-to-one Langmuir combination model ( Evaluation Software version 3.2) Calculate the association rate (k _on ) and dissociation rate (k _off ) by simultaneously fitting the association and dissociation sensorgrams. Equilibrium dissociation constants (Kd) were calculated as the ratio k _off /k _on . See, eg, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the rate of incorporation exceeds 10 ⁶ M ^-1 S ^-1 as determined by surface plasmon resonance above, then the rate of incorporation can be determined using fluorescence quenching techniques, i.e., according to a spectrometer such as a spectrophotometer equipped with a flow cut-off device. (Aviv Instruments) or 8000 series SLM-AMINCO ^TM spectrophotometer (ThermoSpectronic) with stirring cuvette measurements, in the presence of increasing concentrations of antigen, measuring 20nM anti-antigen antibody (Fab format) in PBS pH7.2 ) at 25°C with an increase or decrease in fluorescence emission intensity (excitation=295nm; emission=340nm, 16nm bandpass).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') ₂ , Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see e.g. Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO93/16185; and US Patent Nos. 5,571,894 and 5,587,458. See US Patent No. 5,869,046 for a discussion of Fab and F(ab') ₂ fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives.

Diabodies are antibody fragments that have two antigen-combining sites, which can be bivalent or bispecific. See eg EP404,097; WO1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see eg, US Patent No. 6,248,516 B1 ).

Antibody fragments can be produced by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production of recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, eg, in US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody comprises non-human variable regions (eg, variable regions derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and human constant regions. In yet another example, a chimeric antibody is a "class-switched" antibody, wherein the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, eg, CDRs (or portions thereof) are derived from non-human antibodies and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which HVR residues are derived), eg, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting) ; Padlan, Mol. Immunol.28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR reshuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using "best-fit" methods (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); from Framework regions derived from the consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol. , 151:2623 (1993)); Human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and by screening FR library derived framework regions (see eg Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

4. Human Antibody

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. In general, human antibodies are described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have typically been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, US Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE ^™ technology; US Patent No. 5,770,429, which describes technology; U.S. Patent No. 7,041,870, which describes technology, and U.S. Patent Application Publication No. US2007/0061900, which describes technology). Human variable regions from intact antibodies produced by such animals can be further modified, eg, by combining with different human constant regions.

Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (see, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) -human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20 (3): 927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27 (3): 185 -91 (2005).

Human antibodies can also be generated by isolating variable domain sequences of Fv clones selected from human-derived phage display libraries. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies possessing the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (eds. O'Brien et al., Human Press, Totowa, NJ, 2001), and further described, e.g., in McCafferty et al., Nature 348:552-554; Clackson et al. Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo eds, Human Press, Totowa, NJ , 2003); Sidhu et al., J.Mol.Biol.338(2):299-310(2004); Lee et al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library that can then be screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need for construction of hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies against a large array of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al., EMBO J, 12:725-734( 1993) described. Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and rearranging in vitro using PCR primers containing random sequences encoding the highly variable CDR3 region, as described by Hoogenboom and Winter , J. Mol. Biol., 227:381-388 (1992) described. Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. /0292936 and 2009/0002360.

An antibody or antibody fragment isolated from a human antibody library is considered a human antibody or human antibody fragment herein.

6. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, eg, bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for FGFR4 and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind two different epitopes of FGFR4. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing FGFR4. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO93/08829 , and Traunecker et al., EMBO J. 10:3655 (1991 )), and "protrusion-into-cavity" engineering (see eg US Patent No. 5,731,168). Cross-linking of two or more antibodies or fragments can also be achieved through engineered electrostatic manipulation for generating antibody Fc-heterodimer molecules (WO2009/089004A1) (see e.g. U.S. Patent No. 4,676,980, and Brennan et al. Science, 229:81 (1985)); use leucine zippers to generate bispecific antibodies (see e.g. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); use for the generation of bispecific antibodies "Diabody" technology of specific antibody fragments (see e.g. Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); Gruber et al., J. Immunol., 152:5368 (1994)); and prepare trispecific antibodies to generate multispecific antibodies as described, eg, in Tutt et al. J. Immunol. 147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see eg US2006/0025576A1).

Antibodies or fragments herein also include "dual acting FAbs" or "DAFs" comprising an antigen binding site that binds FGFR4 and another, different antigen (see eg US2008/0069820).

7. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct so long as the final construct possesses the desired characteristics, eg, antigen binding.

a) Substitution, insertion, and deletion variants

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading "Conservative substitutions". More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into an antibody of interest, and the products screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

Amino acids can be grouped according to common side chain properties as follows:

(1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues affecting chain orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions would entail substituting a member of one of these classes for another.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variant selected for further study will have some altered (e.g. improved) biological property relative to the parent antibody (e.g. increased affinity, reduced immunogenicity) and/or will substantially retain the parent antibody certain biological properties. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity).

Changes (eg, substitutions) can be made to the HVR, eg, to improve antibody affinity. HVR "hotspots", residues encoded by codons that undergo mutations at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or Such changes are made in the SDR (a-CDR), wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection has been described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (eds. O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into variable genes selected for maturation by a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed approach, in which several HVR residues (eg, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (eg, conservative substitutions, as provided herein) can be made to HVR that do not substantially reduce binding affinity. Such changes may be outside the HVR "hot spot" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or contains no more than 1, 2 or 3 amino acid substitutions.

One method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and neutralized or negatively charged amino acids (e.g., alanine acid or polyalanine) to determine whether antibody–antigen interaction is affected. Further substitutions may be introduced at amino acid positions showing functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex is used to identify contact points between the antibody and the antigen. As an alternative candidate, such contact residues and neighboring residues can be targeted or eliminated. Variants can be screened to determine whether they contain desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N- or C-terminus of the antibody to an enzyme (eg, for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

b) Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

Where the antibody comprises an Fc region, the carbohydrate to which it is attached can be altered. Native antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides attached, typically via an N-linkage, to Asn297 of the CH2 domain of the Fc region. See, eg, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, hybrid, and high-mannose structures), as determined by Measured by MALDI-TOF mass spectrometry, eg as described in WO2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 (Eu numbering of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ± ± upstream or downstream of position 297 due to minor sequence variations in the antibody 3 amino acids, ie between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, eg, US Patent Publication Nos. US2003/0157108 (Presta, L.); US2004/0093621 (KyowaHakko Kogyo Co., Ltd). Examples of publications dealing with "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108; WO2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US2004/ 0093621;US2004/0132140;US2004/0110704;US2004/0110282;US2004/0109865;WO2003/085119;WO2003/084570;WO2005/035586;WO2005/035778;WO2005/053742;WO2002/031140;Okazaki等J.Mol.Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing afucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. , Presta, L; and WO2004/056312A1, Adams et al., especially in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see for example Yamane- Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Further provided are antibody variants having bisected oligosaccharides, eg, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, eg, in WO2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et al); and US2005/0123546 (Umana et al). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, eg, in WO1997/30087 (Patel et al.); WO1998/58964 (Raju, S.); and WO1999/22764 (Raju, S.).

c) Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. Fc region variants may comprise a human Fc region sequence (eg, a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants that possess some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important and certain Effector functions such as complement and ADCC are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be performed to confirm reduction/ablation of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of molecules of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., the ACTI ^™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind C1q, and thus lacks CDC activity. See eg C1q and C3c binding ELISAs in WO2006/029879 and WO2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see eg Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with two or more substitutions in amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" in which residues 265 and 297 are replaced by alanine Fc mutants (US Patent No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056; WO2004/056312, and Shields et al., J. Biol. Chem. 2001)).

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, eg, substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, changes are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551 , WO99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with prolonged half-life and improved binding to neonatal Fc receptors (FcRn) are described in US2005/0014934A1 (Hinton et al.), responsible for the transfer of maternal IgG to the fetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Those antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, for example, substitutions of Fc region residues 826, 434 7, 1, 7, 7, 7, 434.

See also Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO94/29351, which focuses on other examples of Fc region variants.

d) Antibody variants engineered with cysteine

In certain embodiments, it may be desirable to create cysteine-engineered antibodies, eg, "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In specific embodiments, alternative residues are present at accessible sites of the antibody. By replacing those residues with cysteines, reactive thiol groups are thus positioned in accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to Immunoconjugates were created as described further herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and of the Fc region of the heavy chain S400 (EU numbering method). Antibodies engineered with cysteine can be produced as described, eg, in US Patent No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable modules for antibody derivatization include, but are not limited to, water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 ,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (eg glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under a given condition, etc. .

In another embodiment, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moieties are carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies can be produced using recombinant methods and compositions, eg, as described in US Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-FGFR4 antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of the antibody and/or amino acid sequences comprising the VH (eg, the light and/or heavy chains of the antibody). In yet another embodiment, one or more vectors (eg, expression vectors) comprising such nucleic acids are provided. In yet another embodiment, host cells comprising such nucleic acids are provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) A first vector comprising a nucleic acid encoding an amino acid sequence comprising VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising VH of an antibody. In one embodiment, the host cell is eukaryotic, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (eg, YO, NSO, Sp20 cells). In one embodiment, there is provided a method of producing an anti-FGFR4 antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expression of the antibody, as provided above, and optionally, from the host cell (or host cell culture medium) to recover the antibody.

For recombinant production of anti-FGFR4 antibodies, antibody-encoding nucleic acids (eg, as described above) are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced (eg, by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody) using conventional procedures.

Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli.)). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including those whose glycosylation pathways have been "humanized", resulting in production with partially or fully human glycosylation Patterns of antibodies against fungal and yeast strains. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used with insect cells, in particular for transfecting Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, eg, US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (which describe the PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); Vero cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; buffalo rat) liver cells (BRL3A); human lung cells (W138); Hepatocytes (Hep G2); Mouse Mammary Tumor (MMT060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC5 cells; and FS4 cells. Other useful Examples of mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. B.K.C. Lo, Humana Press, Totowa, NJ), pp. 255- 268 pages (2003).

C. Assay

The anti-FGFR4 antibodies provided herein can be identified, screened for, or characterized for their physical/chemical properties and/or biological activity by a variety of assays known in the art.

1. Binding Assays and Other Assays

In one aspect, the antibodies of the present invention are tested for their antigen-binding activity, for example, by known methods such as ELISA or Western blotting.

In another aspect, competition assays can be used to identify antibodies that compete with antibody LD1 for binding to FGFR4. In certain embodiments, such competing antibodies bind the same epitope (eg, a linear or conformational epitope) that antibody LD1 binds. Detailed exemplary methods for mapping epitopes bound by antibodies are found in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competition assay, in a solution comprising a first labeled antibody (which binds to FGFR4, such as antibody LD1) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to FGFR4) Immobilized FGFR4 was incubated in medium. Secondary antibodies can be present in hybridoma supernatants. As a control, immobilized FGFR4 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the primary antibody to FGFR4, excess unbound antibody was removed and the amount of label associated with immobilized FGFR4 was measured. If the amount of label associated with immobilized FGFR4 is substantially reduced in the test sample compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to FGFR4. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assay

In one aspect, assays for identifying biologically active anti-FGFR4 antibodies are provided. Biological activities may include, for example, inhibition of: FGF (eg, FGF1) stimulated proliferation of FGFR4-expressing cells (eg, HUH7 cells), FGF19-mediated inhibition of CYP7α7 expression in cells exposed to FGF19, FGF19-induced inhibition of FGF19-exposed Phosphorylation of FGFR4, MAPK, FRS2, and/or ERK2 in cells, and FGF19-induced colony formation (in some embodiments, colony formation by HCC cell lines).

Also provided are antibodies having such biological activity in vivo and/or in vitro.

In certain embodiments, antibodies of the invention are tested for such biological activities. Assays for testing such biological activities are described herein.

In certain embodiments, antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibition of cell growth or proliferation are well known in the art. Certain assays of cell proliferation, exemplified by the "cell killing" assays described herein, measure cell viability. One such assay is the CellTiter-Glo ^™ Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on the quantification of the ATP present, an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-88; US Patent No. 6,602,677. The assay can be performed in 96-well or 384-well format, making it amenable to automated high-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs 6:398-404. The assay procedure involves adding a single reagent directly to cultured cells ( reagents). This results in cell lysis and generation of a luminescent signal by the luciferase reaction. The luminescence signal is directly proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by photometer or CCD camera imaging device. Luminescent output is expressed in relative light units (RLU).

Another assay for cell proliferation is the "MTT" assay, a method that measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide by mitochondria Enzymatic oxidation to formazan (formazan) colorimetric assay. Like the CellTiter-Glo ^™ assay, this assay indicates the number of metabolically active cells present in the cell culture. See, eg, Mosmann (1983) J. Immunol. Meth. 65:55-63; and Zhang et al. (2005) Cancer Res. 65:3877-3882.

Cells for use in any of the above in vitro assays include cells or cell lines that naturally express FGFR4 or have been engineered to express FGFR4. Such cells include tumor cells that overexpress FGFR4 relative to normal cells of the same tissue origin. Such cells also include cell lines expressing FGFR4 (including tumor cell lines) and cell lines that do not normally express FGFR4 but have been transfected with a nucleic acid encoding FGFR4. Exemplary cell lines provided herein for use in any of the above in vitro assays include the HCC cell line HUH7.

In one aspect, anti-FGFR4 antibodies of the invention are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, anti-FGFR4 antibodies of the invention are tested for their ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such testing. In one exemplary xenograft system, human tumor cells are introduced into an appropriately immunocompromised non-human animal, such as an athymic "nude" mouse. An antibody of the invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the aforementioned xenograft systems, the human tumor cells are tumor cells from a human patient. Such xenograft models are commercially available from Oncotest GmbH (Frieberg, Germany). In certain embodiments, the aforementioned human tumor cells are cells from a human tumor cell line, such as HUH7 HCC tumor cells. In certain embodiments, human tumor cells are introduced into an appropriately immunocompromised non-human animal by subcutaneous injection or by transplantation into a suitable site, such as the mammary fat pad. In certain embodiments, the xenograft model is a transgenic mouse overexpressing FGF19, such as described herein and in nicholes et al, Am J Pathol 160:2295-2307.

It is understood that any of the above assays may be performed using the immunoconjugates of the invention in place of or in addition to anti-FGFR4 antibodies.

It is understood that any of the above assays can be performed using anti-FGFR4 antibodies and other therapeutic agents, such as chemotherapeutic agents.

D. Immunoconjugates

The present invention also provides a combination comprising one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (such as protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or Radioisotope-conjugated immunoconjugates of the anti-FGFR4 antibodies herein.

In another embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Patent No. 5,208,020, 5,416,064 and European Patent EP0 425 235B1); auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); Calicheamicin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342); and (1Lo93 , Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al., CurrentMed.Chem.13:477-523 (2006 ); Jeffrey et al, Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al, Bioconj. Chem. (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J.Med.Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methylamine pterins; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin, or a fragment thereof, including but not limited to diphtheria A chain, non-binding active fragments of diphtheria toxin , exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α - Inhibition of sarcin, Aleutites fordii toxin, dianthin toxin, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia Curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin , phenomycin, enomycin and trichothecenes.

In one embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the generation of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and radioactive isotopes of Lu. When radioconjugates are used for detection, it can contain radioactive atoms such as tc99m or I123 for scintillation studies, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri) substances such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

A variety of bifunctional protein coupling agents can be used to generate conjugates of antibodies and cytotoxic agents, such as N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl -4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), imidate (such as dimethyl adipimidate hydrochloride esters), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azides (such as bis(p-azidobenzoyl)hexamethylenediamine), double Nitrogen derivatives (such as bis(p-diazobenzoyl)-ethylenediamine), diisothiocyanates (such as toluene 2,6-diisocyanate), and bis-reactive fluorine compounds (such as 1,5-di Fluoro-2,4-dinitrobenzene) bifunctional derivatives. For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile, peptidase-sensitive, photolabile, dimethyl, or disulfide-containing linkers can be used (Chari et al., Cancer Res 52:127-131 (1992); U.S. Patent No. 5,208,020) .

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl -(4-vinylsulfone)benzoate), which are commercially available (eg, from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

E. Methods and Compositions for Diagnosis and Detection

In certain embodiments, any of the anti-FGFR4 antibodies provided herein can be used to detect the presence of FGFR4 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as breast/breast, pancreas, esophagus, lung and/or brain.

In one embodiment, an anti-FGFR4 antibody for use in a method of diagnosis or detection is provided. In yet another aspect, methods of detecting the presence of FGFR4 in a biological sample are provided. In certain embodiments, the method comprises contacting a biological sample with an anti-FGFR4 antibody under conditions permissive for binding of the anti-FGFR4 antibody to FGFR4, as described herein, and detecting whether a complex is formed between the anti-FGFR4 antibody and FGFR4 things. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-FGFR4 antibody is used to select subjects suitable for treatment with an anti-FGFR4 antibody, eg, wherein FGFR4 is a biomarker for patient selection.

Exemplary disorders that can be diagnosed using the antibodies of the invention include cancer (e.g., breast cancer, lung cancer, pancreatic cancer, brain cancer, kidney cancer, gastric cancer, leukemia, endometrial cancer, colon cancer, prostate cancer, pituitary cancer, breast fibrosis adenoma, head and neck cancer, soft tissue cancer, neuroblastoma, melanoma, endometrial cancer, testicular cancer, bile duct cancer, gallbladder cancer, and liver cancer).

In certain embodiments, labeled anti-FGFR4 antibodies are provided. Labels include, but are not limited to, labels or moieties for direct detection (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and moieties for indirect detection, such as via enzymatic reactions or molecular interactions, such as enzymes or ligands. body. Exemplary labels include, but are not limited to, radioactive isotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, Horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6 - Phosphate dehydrogenases, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to enzymes such as HRP that employ hydrogen peroxide to oxidize dye precursors), lactoperoxidase, or microperoxidase , biotin/avidin, spin tags, phage tags, stable free radicals, and more.

F. Pharmaceutical formulations

Prepared as lyophilized formulations or aqueous solutions by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Ed., Osol, A. Ed. (1980)). Pharmaceutical formulations of anti-FGFR4 antibodies as described herein. In general, pharmaceutically acceptable carriers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and formazan; Thiamine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexanediamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; paraben Hydrocarbyl esters such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine ; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metals complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further comprise interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO2006/044908, the latter formulation comprising a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an EGFR antagonist (such as erlotinib), an anti-angiogenic agent (such as a VEGF antagonist, such as an anti-VEGF antibody), or a chemotherapeutic agent (such as taxanes or platinum agents). Such active ingredients are suitably present in combination in amounts effective for the desired purpose.

The active ingredient can be entrapped, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization (such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th Ed., Osol, A. Ed. (1980).

Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody in shaped commercial forms, such as films, or microcapsules.

Formulations for in vivo administration are generally sterile. Sterility is readily achieved, for example, by filtration through sterile filters.

G. Therapeutic Methods and Compositions

Any of the anti-FGFR4 antibodies provided herein can be used in therapeutic methods.

In one aspect, anti-FGFR4 antibodies for use as a medicament are provided. In other aspects, anti-FGFR4 antibodies for use in treating cancer are provided. In other aspects, anti-FGFR4 antibodies for use in treating liver disease (eg, cirrhosis) are provided. In other aspects, anti-FGFR4 antibodies for use in treating a wasting disorder are provided. In certain embodiments, anti-FGFR4 antibodies for use in methods of treatment are provided. In certain embodiments, the invention provides an anti-FGFR4 antibody for use in a method of treating an individual with cancer, the method comprising administering to the individual an effective amount of an anti-FGFR4 antibody. In certain embodiments, the invention provides an anti-FGFR4 antibody for use in a method of treating an individual having a liver disorder, such as cirrhosis, the method comprising administering to the individual an effective amount of the anti-FGFR4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. In yet other embodiments, the invention provides anti-FGFR4 antibodies for use in inhibiting cell proliferation.

In certain embodiments, the invention provides anti-FGFR4 antibodies for use in a method of inhibiting cell proliferation in an individual, the method comprising administering to the individual an effective amount of the anti-FGFR4 antibody to inhibit cell proliferation. An "individual" according to any of the above embodiments is preferably a human.

In yet another aspect, the present invention provides the use of an anti-FGFR4 antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat cancer. In yet another embodiment, the medicament is for use in a method of treating cancer comprising administering an effective amount of the medicament to an individual having cancer. In one embodiment, the medicament is for the treatment of a liver disorder (such as cirrhosis, non-alcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis). In yet another embodiment, the medicament is for use in a method of treatment of a liver disorder (such as cirrhosis, nonalcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis), comprising the treatment of An individual with a hepatic disorder is administered an effective amount of the drug. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, eg, as described below. In yet another embodiment, the drug is used to inhibit cell proliferation. In yet another embodiment, the medicament is for use in a method of inhibiting cell proliferation in an individual, the method comprising administering to the individual an effective amount of the medicament to inhibit cell proliferation. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides methods of treating cancer. In one embodiment, the method comprises administering to an individual having such cancer an effective amount of an anti-FGFR4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the present invention provides methods of treating liver disorders such as cirrhosis, nonalcoholic fatty liver disease, biliary cirrhosis, sclerosing cholangitis, progressive familial intrahepatic cholestasis. In one embodiment, the method comprises administering to an individual having such a liver disorder an effective amount of an anti-FGFR4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides methods for treating a wasting disorder. In one embodiment, the method comprises administering to an individual having such a wasting disorder an effective amount of an anti-FGFR4 antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. An "individual" according to any of the above embodiments may be a human.

In yet another aspect, the invention provides methods for inhibiting cell proliferation in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-FGFR4 antibody to inhibit cell proliferation. In one embodiment, an "individual" is a human.

In yet another aspect, the invention provides a pharmaceutical formulation comprising any of the anti-FGFR4 antibodies provided herein, eg, for use in any of the aforementioned therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the anti-FGFR4 antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the anti-FGFR4 antibodies provided herein and at least one other therapeutic agent, eg, as described below.

Antibodies of the invention may be used alone or in combination with other agents in therapy. For example, an antibody of the invention can be co-administered with at least one other therapeutic agent. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent.

Such combination therapy noted above encompasses combined administration (where two or more therapeutic agents are contained in the same or different formulations), and separate administration, in which case it may be administered in combination with other therapeutic agents and/or Administration of the antibody of the invention occurs before, simultaneously with, and/or after the adjuvant. Antibodies of the invention may also be used in combination with radiation therapy.

Antibodies of the invention (and any other therapeutic agent) may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether the administration is transient or chronic, dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection. Various dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

The antibodies of the invention should be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular condition being treated, the particular mammal being treated, the clinical state of the individual patient, etiology, site of drug delivery, method of administration, dosing schedule, and other factors known to the practitioner. Antibodies need not, but can optionally, be formulated with one or more drugs currently used to prevent or treat the disorder. Effective amounts of the other agents described above depend on the amount of antibody present in the formulation, the type of condition being treated, and other factors discussed above. These drugs are generally used in the same dosage and with the route of administration described herein, or at about 1-99% of the dosage described herein, or in any dosage and by any route, said dosage and route are Empirically determined/clinically determined to be appropriate.

In order to prevent or treat diseases, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) should depend on the type of disease to be treated, the type of antibody, and the severity of the disease and the course of the disease, the prophylactic or therapeutic purpose of the antibody administered, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. Antibodies are suitable for administration to a patient at one time or in a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg-15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of the antibody may be administered to the patient as the first candidate dose, either e.g. by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment should generally be continued until the desired suppression of the condition occurs. Such doses may be administered intermittently, such as once every week or every three weeks (eg such that the patient receives from about 2 to about 20, or eg about 6 doses of the antibody). An initial higher loading dose, followed by one or more lower doses, may be given. However, other dosing regimens can be used. The progress of this therapy is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above formulations or therapeutic methods may be practiced using the immunoconjugates of the invention in place of and in addition to anti-FGFR4 antibodies.

H. Products

In another aspect of the invention there is provided an article of manufacture comprising materials useful for the treatment, prevention and/or diagnosis of the disorders described above. Articles of manufacture comprising containers and labels or package inserts on or associated with containers. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition effective to treat, prevent, and/or diagnose a condition, alone or in combination with another composition, and can have a sterile access opening (e.g., the container can be a tube with a stopper passable by a hypodermic needle vial or bag of intravenous solution). At least one active agent in the composition is an antibody of the invention. The label or package insert directs use of the composition to treat the condition of choice. Additionally, the article of manufacture may comprise (a) a first container having a composition contained therein, wherein the composition comprises an antibody of the invention; and (b) a second container having a composition contained therein, wherein the composition comprises additional cells Toxic or otherwise therapeutic agents. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further contain other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It should be understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-FGFR4 antibody.

III. Example

A. Anti-FGFR4 antibody treatment inhibits hepatocellular carcinoma

Materials and methods

In silico expression analysis. For expression analysis, box-plots and whisker-plots were generated for FGFR4 using normalized gene expression data extracted from the BioExpress ^™ database (Gene Logic, Gaithersburg, MD). The distribution of FGFR4 expression in normal and cancerous tissues was assessed using the signal associated with probe 204579_at.

immunochemistry. Formalin-fixed, paraffin-embedded tissue sections were processed for antigen retrieval using Trilogy (Cell Marque, Rocklin, CA) and incubated with 10 μg/ml anti-FGFR4 antibody (8G11; Genentech, South San Francisco, CA). Immunostaining was achieved using a biotinylated secondary antibody ABC-HRP reagent (Vector Laboratories, Burlingame, CA) and a metal-enhanced DAB colorimetric peroxidase substrate (Thermo Fisher Scientific, Rockford, IL).

Semi-quantitative RT-PCR. Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA). Gene expression is amplified and quantified using specific primers and fluorescent probes (31). Gene-specific signals were normalized to the RPL19 housekeeping gene. All TaqMan qRT-PCR reagents were purchased from AppliedBiosystems (Foster City, CA). A minimum of three sets of data were analyzed for each condition. Data are expressed as mean ± SEM.

Immunoprecipitation and immunoblotting. Supplemented with complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN), phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich, St.Louis, MO), 2mM sodium fluoride, and 2mM sodium orthovanadate Lysates of cultured cells or frozen tissues were prepared with RIPA Lysis Buffer (Millipore, Billerica, MA). BCA assay (Thermo The equivalent amount of protein determined by Fisher Scientific). For human liver lysates, immunoblot analysis was preceded by FGFR4 immunoprecipitation as previously described (16).

Generation of FGFR4 monoclonal antibody. FGFR4-null mice were immunized with recombinant human and mouse FGFR4-Fc chimeric proteins (Genentech). Spleens were collected 8 weeks later and hybridomas were generated. Culture supernatants were collected and screened against the immunogen by a solid phase antibody binding assay. Positive cell lines were further screened for their efficacy in inhibiting FGF1 and FGF19 binding to human and mouse FGFR4 using a solid phase receptor binding assay. LD1 producing hybridomas were subcloned twice to ensure monoclonality.

Molecular cloning of LD1. Total RNA was extracted from muLD1-producing hybridoma cells using the RNeasy Mini Kit (Qiagen). The light and heavy chain variable domains were amplified using reverse transcription-PCR (RT-PCR). The forward primer is specific for the NH2-terminal amino acid sequence of the light chain variable region and the heavy chain variable region. Light chain and heavy chain reverse primers were designed to anneal to regions of the light chain constant domain and heavy chain first constant domain that are highly conserved across species, respectively. The amplified light chain variable domains were cloned into mammalian expression vectors containing human kappa constant domains. The amplified heavy chain variable domain was inserted into a mammalian expression vector encoding the full length human IgG1 constant domain. Chimeric antibodies were transiently expressed as previously described (16). The experiments described in this Part A of the Examples (including Figures 1-9) used chimeric LD1.

Solid Phase Antibody Binding Assay. Maxisorp 96-well plates were coated with 50 μl 2 μg/ml anti-human immunoglobulin Fc fragment specificity (Jackson ImmunoResearch Laboratories, West Grove, PA) or anti-FLAG antibody (Sigma-Aldrich) overnight at 4°C. Nonspecific binding sites were saturated with 200 μl PBS/3% bovine serum albumin (BSA) for 1 hr, and FGFR-IgG (Genentech and R&D Systems, Minneapolis, MN) or FLAG-tagged FGFR4 in PBS/0.3% BSA (FGFR4ΔTM-FLAG) were incubated for 1 hr. Plates were washed and incubated with anti-FGFR4 antibody in PBS/0.3% BSA for 1 hr. Bound antibodies were detected using HRP-conjugated anti-IgG (Jackson ImmunoResearch Laboratories) and TMB peroxidase chromogenic substrate (KPL, Gaithersburg, MD).

Flow cytometry analysis. Cells for flow cytometry analysis were resuspended in PBS containing 5 mM EDTA and washed with PBS containing 2% heat-inactivated fetal bovine serum (FBS). Perform all subsequent steps on ice. Cells ( ^1x106 ) were incubated with primary antibody (LD1 or isotype control) for 30 min, followed by incubation with phycoerythrin (PE)-conjugated anti-human IgG antibody (Jackson ImmunoResearch). Cells were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA).

DNA constructs. Human FGFR4 (hFGFR4) cDNA was cloned as previously described (16). The ectodomain of FGFR4 was also subcloned into the expression vector pCMV-Tag4A (Stratagene, La Jolla, CA) to obtain a secreted form of FGFR4 with a FLAG tag at the C-terminus (FGFR4ΔTM-Flag). Single-nucleotide mutations were introduced in the FGFR4ΔTM-Flag construct using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). We also generated a human FGFR4FGFR1 chimeric construct comprising the fused extracellular and transmembrane domains of human FGFR4 (amino acid residues M1-G392 of FGFR4) and the cytoplasmic domain of human FGFR1 (amino acid residues K398-R820 of FGFR1) ( hFGFR4/R1). The amino acid sequence linking FGFR4 (bold) and FGFR1 ( normal ) is . . . AVLLLLAGLYRG KMKSG . (SEQ ID NO: 32) . The hFGFR4 cDNA or hFGFR4/R1 cDNA was ligated into the pQCXIP retroviral bicistronic expression vector (Clontech Laboratories, Mountain View, CA).

FGFR4ΔTM-Flag conditioned medium. HEK293 cells were transfected with wild-type or mutant FGFR4ΔTM-Flag constructs or corresponding empty vectors and maintained in serum-free PS25 medium for 72 to 96 hours. The resulting medium was filtered, supplemented with HEPES pH 7.2 (final concentration 40 mM) and protease inhibitors, and stored at 4°C until use.

Cell culture and stable cell lines. HEK293, HEPG2, and HEP3B cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in a F12:DMEM mixture (50:50) supplemented with 10% FBS and 2 mM L-glutamine. HUH7 and PLC/PRF/5 cells were cultured in DMEM high glucose, 10% FBS. JHH4, JHH5, and JHH7 cells were purchased from the Japanese Cancer Research Resource Bank (Tokyo, Japan) and maintained in Williams medium E supplemented with 10% FBS and 2 mM L-glutamine. SNU449 cells were obtained from ATCC and maintained in RPMI1640 containing 10% FBS and 2mM L-glutamine. BaF3 cells were maintained in RPMI1640 (Life Technologies, Carlsbad, CA) supplemented with 10% FBS, 1 ng/ml IL-3, and 2 mM L-glutamine. L6 cells were obtained from ATCC and maintained in DMEM high glucose supplemented with 10% FBS.

Infect cultures of BaF3 and L6 cells with empty, hFGFR4, or hFGFR4/R1 retroviral expression vectors following the manufacturer's recommendations and select for 10 to 12 days in media containing 2.5 μg/mL puromycin (Life Technologies) . The fifth percentile highest expressing cells were isolated from selected pools by fluorescence activated cell sorting (FACS) using an anti-FGFR4 antibody (8G11; Genentech). The resulting collection of cells expressing high levels of FGFR4, high levels of FGFR4/R1 and control cells stably transfected with empty vector were maintained in complete medium containing 2.5 μg/mL puromycin.

Mitogenic Assay. BaF3/control, BaF3/FGFR4, and BaF3/FGFR4/R1 cells were washed twice and seeded in 96-well plates (22,500 cells/well). FGF was added to each well, and cells were incubated at 37°C for 72 hours. Relative cell density was measured using CellTiter Glo (Promega, Madison, WI) following the manufacturer's recommendations.

Inhibition of FGF pathway activation by anti-FGFR4 antibody. Cells were serum starved for 24 hours in the absence or presence of LD1 or isotype control antibody. They were then stimulated with 5 ng/ml FGF1 (FGF acidic, R&D Systems) and 10 μg/ml heparin for 5 min. Cells were lysed with RIPA lysis buffer (Millipore) supplemented with complete EDTA-free protease inhibitor cocktail (Roche), phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich), 2 mM sodium fluoride, and 2 mM sodium orthovanadate. Equal amounts of protein were analyzed by immunoblotting using antibodies against phospho-ERK1/2, phospho-FRS2, ERK1/2 (Cell Signaling Technology, Danvers, MA), and FRS2 (Millipore).

Clonogenic Assay. Seed HUH7 (5,000 cells/well), PLC/PRF/5 (2,000 cells/well), JHH5 (500 cells/well), or JHH5/hFGFR4 shRNA (500 cells/well) cells in triplicate. HUH7 and PLC/PRF/5 cells were treated with or without anti-FGFR4 antibody (chLD1; Genentech) 3 hr after seeding. Antibodies were replaced twice a week during the experiment (14 days). For JHH5 and JHH5/hFGFR4 shRNA cells, treatment was initiated with or without 2 mg/ml doxycycline 3 hr after seeding and changed three times a week during the experiment. Cells were washed with PBS and stained with 0.5% crystal violet solution. Colonies were counted using MetaMorph software (Molecular Devices, Sunnyvale, CA).

In vivo experiments. All animal protocols were approved by the Research Animal Care and Use Committee. Female nu/nu mice aged 5 to 6 weeks were obtained from Charles River Laboratories International (Wilmington, MA). Mice were provided with standard chow and water ad libitum until 12 hours before injection, at which time the chow was removed. Mice were given intraperitoneal (IP) injection (10 mg/kg) of control or anti-FGFR4 (chLD1) antibody. After 18 h, mice received vehicle (PBS) or 1 mg/kg FGF19 intravenously (IV). After 30 min, mice from all groups were necropsied and tissue samples collected, frozen in liquid nitrogen, and stored in -70°C. Total RNA from frozen tissue samples was prepared using the RNeasy kit (Qiagen). Each condition was analyzed on groups of 3 to 5 animals. Data are expressed as mean ± SEM and analyzed by Student's t-test.

For xenograft experiments, 6 to 8 week old nu/nu female mice (Charles River Laboratories International) were inoculated subcutaneously with ^5x106 cells (200ul/mouse) and Matrigel (BD Biosciences). After 7 days, mice bearing tumors of equivalent volume (~150 mm ³ ) were randomized into groups (n=10) and treated IP twice a week. Tumors were measured with electronic calipers (Fowler Sylvac Ultra-Cal MarkIII; Fred V. Fowler Company, Newton, MA) and the mean tumor volume was calculated using the formula (W ² x L)/2, where W and L were the smaller diameter and larger diameter. Data are expressed as mean tumor volume ± SEM and analyzed by Student's t-test.

FGF19 transgenic mice were generated as previously described (32). FGFR4 null mutant (FGFR4-KO) animals were constructed as previously reported (33) and were provided by W.L. McKeehan under a material transfer protocol (University of Texas Southwestern Medical Center, Dallas, TX). Mice that both overexpress FGF19 and lack the FGFR4 receptor (FGF19-TG:FGFR4-KO) were generated by crossing young adult FGF19-TG males with young adult FGFR4-KO females. The presence of two genetic engineering events was confirmed by tail DNA PCR at weaning.

result

FGFR4 is required for hepatocarcinogenesis in FGF19 transgenic mice. It was previously shown that exogenous expression of FGF19 in transgenic mice caused HCC by 10 months of age (18). To assess whether FGFR4 is involved in this FGF19-mediated tumorigenesis, we mated FGF19 transgenic (FGF19-TG) mice with FGFR4 knockout (FGFR4-KO) mice or FGFR4 wild-type (FGFR4-WT) mice. Mice were necropsied at various time points and hepatocarcinogenesis was assessed by performing macroscopic and histological examination and by measuring preneoplastic hepatocyte proliferation (ie, BrdU incorporation). The development of HCC in FGF19-TG:FGFR4-WT mice was previously described (18). In contrast to FGF19-TG:FGFR4-WT mice, FGF19-TG:FGFR4-KO mice did not develop macroscopic or histological evidence of hepatoma formation at any time during this experiment (Fig. 1A). Also, preneoplastic hepatocyte proliferation was significantly elevated in FGF19-TG mice with the FGFR4-WT genotype, but not in FGF19-TG:FGFR4-KO littermates (Fig. 1B). Consistent with previous reports of higher frequency and severity of tumors in female FGF19-TG mice (18), BrdU incorporation was increased in FGF19-TG:FGFR4-WT females compared to corresponding males (compare Figure 1B left and small picture on the right). We also assessed the effect of the potent hepatocarcinogen diethylnitrosamine (DEN) on HCC development in FGF19-TG mice. Administration of DEN accelerated HCC development in FGF19-TG:FGFR4-WT mice. A full range of tumors was seen by 4 months of age in livers from all DEN-treated FGF19-TG:FGFR4-WT animals compared to 10 months of age for DEN-untreated FGF19-TG:FGFR4-WT mice Pre- and neoplastic lesions – altered (basophilic) hepatic foci, pericentric hepatocyte dysplasia, well-differentiated hepatoma, and aggressive hepatocellular carcinoma (Fig. 1D). The major morphological feature of livers from nearly all FGF19-TG:FGFR4-WT mice at all time points was macroscopically apparent HCC nodules on multiple lobes (Fig. 1C). Tumor burden was assessed by measuring liver weight. Relative liver weight gradually increased at all time points in FGF19-TG:FGFR4-WT mice treated with DEN (Fig. 1E). Interestingly, the increase in liver weight was more pronounced in females (2.7-fold at 6 months) than in males (1.8-fold at 6 months) (compare left and right panels of Fig. 1E). It should be noted that none of the males survived beyond 6 months of age (Fig. 1E). Hepatocarcinogenesis observed in DEN-treated FGF19-TG:FGFR4-WT mice was abrogated by abrogation of FGFR4 expression in FGFR4-KO mice. Thus, the relative liver weight of FGF19-TG:FGFR4-KO mice remained constant during adulthood (Fig. 1F). These results suggest that FGF19-promoted hepatocarcinogenesis in mice requires FGFR4 expression.

Generation of neutralizing monoclonal antibodies against FGFR4. To assess whether targeting FGFR4 could have therapeutic impact in HCC, we generated FGFR4-specific monoclonal antibodies by immunizing FGFR4-KO mice with recombinant and human FGFR4. One of the resulting clones (termed LD1) was selected based on its specificity for binding mouse, macaque, and human FGFR4 (Fig. 2A). This antibody does not bind mouse or human FGFR1, FGFR2, or FGFR3 (Figure 2A). Surface plasmon resonance analysis revealed that LD1 bound mouse, macaque, and human FGFR4 with comparable affinity (Fig. 2B). We used flow cytometry to assess whether LD1 binds to FGFR4 present on the cell surface. The specific binding of LD1 to HEK293 cells stably transfected with human FGFR4 was proportional to the added antibody concentration (Fig. 2C). LD1 does not bind to control HEK293 cells stably transfected with empty vector. Together these data demonstrate that LDl specifically binds mouse, macaque, and human FGFR4 and also recognizes the human receptor expressed on the cell surface.

To map the FGFR4 epitope for LD1, we compared the amino acid sequences of mouse and human FGFR1, FGFR2, FGFR3, and FGFR4. Eight amino acids were selected based on their similarity between FGFR4 orthologs and their dissimilarity in FGFR1-3 orthologs. These amino acids in FGFR4 were substituted with amino acids present at equivalent positions in FGFR3 to generate eight different mutant constructs of human FGFR4. These constructs were expressed and LD1 binding assessed using a solid phase binding assay. LD1 bound wild-type FGFR4 and most of the mutant constructs equally well; G165A was the only FGFR4 mutant in which LD1 binding was impaired (Fig. 2D). LD1 did not bind negative control wild-type FGFR3 (Fig. 2D). We also tested LD1 binding to the mutant constructs using immunoblot analysis. All previously described protein constructs were reduced, denatured, electrophoresed, and electrotransferred to nitrocellulose. Nitrocellulose membranes were incubated sequentially with LD1, an anti-FGFR4 antibody (8G11) recognizing different epitopes, or an anti-FLAG antibody. Anti-FLAG antibodies and 8G11 detected wild-type FGFR4 and all FGFR4 mutant constructs, whereas LD1 detected all constructs equally well except the G165A mutant (Fig. 2E). No protein bands were detected by any antibody in the control lane (Fig. 2E). We generated a 3D model of a FGFR4 dimer bound to two molecules of FGF19 to visualize the location of G165 (Fig. 2F). G165 is located in the center of the FGFR4-FGF19 complex, at the point of contact between the two FGFR4 subunits. Together these results show that G165 is critical for the interaction of LD1 with human FGFR4. LD1 binding to reduced and denatured FGFR4 also suggests that the epitope is independent of tertiary conformation.

We next used a solid-phase receptor binding assay to test whether LD1 could block the binding of FGF1 and FGF19 to FGFR4. LD1 inhibited FGF binding in a dose-dependent manner with IC50 reaching 0.093 ± 0.006 nM for FGF1 and 0.102 ± 0.003 nM for FGF19 (Fig. 3A). To assess whether LD1 could inhibit the function of cell surface-expressed FGFR4, we first utilized a BaF3 mouse pre-B cell line stably transfected with a chimeric construct encoding the extracellular domain of FGFR4 and the intracellular domain of FGFR1 (BaF3/FGFR4/R1) (pro-Bcell line). The wild-type BaF3 cell line is an interleukin-3 (IL-3)-dependent cell line that does not express any FGFRs. BaF3 cells transfected with FGFR proliferated in the absence of IL-3 when stimulated with FGF and heparin (21). Transfection of this construct allowed us to replace IL-3 with FGF to support the growth of BaF3 cells. In the presence of 5 nM FGF1, LD1 inhibited the proliferation of BaF3/FGFR4/R1 cells with IC50 of 17.4±5.4 nM (Fig. 3B).

We also used an L6 rat skeletal muscle cell line stably transfected with a vector expressing FGFR4 (L6/FGFR4) to assess the effect of LD1 on FGF signaling. Addition of FGF1 and heparin to L6/FGFR4 cell cultures activates the FGFR pathway, as evidenced by phosphorylation of FGFR substrate 2 (FRS2) and extracellular signal-regulated kinase 1/2 (ERK1/2), whereas LD1 in Ligand-induced phosphorylation of these second messengers was inhibited in a dose-dependent manner (Fig. 3C). Interestingly, addition of LD1 also triggered an increase in total FRS2 content in these cells (Fig. 3C).

Using flow cytometry, we assessed the binding of LD1 and confirmed the expression of FGFR4 on the cell surface of a subset of HCC cell lines. LD1 bound PLC/PRF/5 to the highest extent and to a lesser extent HUH7 and JHH5 cells (Fig. 3D). The binding of the control antibody to the surface of these cells was negligible (Fig. 3D). Moreover, binding of LD1 and control antibodies to the surface of BaF3 cells (which were used as a negative control because they do not express FGFR4) was also negligible (Fig. 3D).

LD1 inhibits FGFR4 function in hepatoma cell lines. The inhibitory activity of LD1 was characterized using hepatoma cell lines with various levels of expression of endogenous FGFRs (ie, FGFR1-4) (Fig. 7). In HEP3B cells, addition of FGF19 triggered phosphorylation of FRS2 and ERK1/2, whereas LD1 inhibited FRS2 phosphorylation stimulated by FGF19 (Fig. 4A), similar to its effect in L6/FGFR4 cells. However, LD1 did not appreciably alter ERK1/2 phosphorylation (Fig. 4A).

Expression of cytochrome P4507α1 (CYP7α1) and c-Fos in hepatic cell lines is regulated by FGF19 (16,22). We tested whether LD1 could repress this FGF19-mediated gene regulation. In HEB3B cells, addition of FGF19 reduced the expression of CYP7α1 by 81% (Fig. 4B). Addition of LD1 restored 67% of basal expression of CYP7α1 (Fig. 4B). LD1 increased CYP7α1 expression by 2-fold in the absence of FGF19 (Fig. 4B). While addition of FGF19 did not affect CYP7α1 expression in HUH7 cells, addition of LD1 had a similar effect as in HEP3B cells, increasing the expression of this gene by 2.9- and 3.5-fold in the presence or absence of FGF19, respectively (Fig. 8). Addition of negative control antibody had no effect on CYP7α1 expression in either HEP3B or HUH7 cells (Figures 4B and 8, respectively). Interestingly, addition of LD1 resulted in upregulation of CYP7α1 expression in both HEP3B and HUH7 cells without exogenous addition of FGFR4 ligand. This indicates that LD1 inhibits basal FGFR4 activity likely maintained through the FGFR4 ligand autocrine/paracrine loop.

To further assess the effect of LD1 on the basal activity of FGFR4, we measured c-Fos expression in the absence of exogenously added FGFR4 ligands. Activation of the FGFR4 pathway was previously shown to increase c-Fos expression (16). Addition of LD1 reduced basal expression of c-Fos by 50% in JHH5, JHH7, and HUH7 cell lines and by 75% in PLC/PRF/5 cell lines; addition of control antibody had no effect on basal c-Fos expression (Fig. 4C ). These results demonstrate the ability of LD1 to inhibit the basal activity of FGFR4.

LD1 inhibits colony formation. We first measured colony formation by JHH5 cells stably transfected with doxycycline-inducible FGFR4-specific shRNA or control shRNA. While JHH5 cells transfected with control constructs did not differ in their ability to form colonies in the absence or presence of doxycycline, addition of doxycycline to constructs with FGFR4shRNA The colony formation was inhibited by 76% in the JHH5 cells transfected with the drug (Fig. 4D). This result suggests that FGFR4 is involved in the colony formation of liver cancer cell lines.

We next tested the ability of LD1 to inhibit colony formation of a panel of hepatoma cell lines. Addition of LD1 to cultures of JHH5, HUH7, and PLC/PRF/5 cells caused a dose-dependent reduction in dose formation, reaching a maximal inhibition of 26%, 50%, and 82%, respectively (Fig. 4F). Figure 4E shows representative examples of PLC/PRF/5 and HUH7 cell cultures. Addition of control antibody did not affect colony formation (Figure 4E and 4F). These results indicate that LD1 inhibits FGFR4-mediated colony formation in hepatoma cell lines.

LD1 inhibits FGFR4 activity in vivo. We assessed the in vivo efficacy of LD1 by measuring FGF19-triggered induction of c-Fos in the liver of mice injected with LD1 or a control antibody. We chose to monitor the c-Fos response to FGF19 because c-Fos induction in the liver is sensitive to FGF19 stimulation (16). c-Fos expression was 53-fold higher in the livers of mice treated with FGF19 compared with those treated with phosphate-buffered saline (PBS) (Fig. 5A). Administration of LD1 18 h before FGF19 injection reduced c-Fos induction by 3.5-fold (Fig. 5A). LD1 also reduced the basal level of c-Fos expression in untreated mice by 6-fold (Fig. 5A). Injection of control antibody did not alter basal or FGF19-stimulated c-Fos expression compared with untreated mice (Fig. 5A). These data demonstrate the in vivo efficacy of LD1 in inhibiting both basal and FGF19-stimulated FGFR4 activity.

LD1 inhibits tumor growth in vivo. To examine the in vivo efficacy of LD1 in suppressing tumor growth, we first utilized the HUH7 hepatoma cell line xenograft model. Mice bearing established tumors (approximately 150 mm ³ ) were dosed weekly with 30 mg/kg LD1, 30 mg/kg control antibody, or PBS. After 13 days, HUH7 tumors from mice treated with either PBS or control antibody grew to an average size of 720 mm ³ ( FIG. 5B ). However, HUH7 tumors in mice treated with LD1 grew to an average size of 28 mm ³ , with a 96% inhibition of tumor growth compared to control antibody or PBS ( FIG. 5B ). In repeated experiments, twice-weekly administration of 30 mg/kg LD1 caused complete tumor growth inhibition (Figure 9). At necropsy, tumors were excised and the effect of LD1 on the expression of FGFR4 and genes regulated by FGFR4 was assessed. Administration of LD1 did not affect FGFR4 expression in HUH7 xenograft tumors (Fig. 5C). However, LD1 increased the expression of CYP7α1 by 3-fold compared with the level of CYP7α1 expression measured in tumors of mice treated with PBS (Fig. 5C). LD1 also decreased the expression of c-Fos and egr-1 by 17- and 6-fold, respectively, compared with mice treated with PBS (Fig. 5C).

To further evaluate the in vivo efficacy of LD1, we used the FGF19-TG mouse model. FGF19-TG mice were treated with DEN at 15 days of age to accelerate tumorigenesis, and then randomized into 3 groups at 4 weeks of age. One group received a control antibody and the other two groups received either LD1 or an anti-FGF19 antibody (1A6) on a weekly basis. 1A6 was previously shown to prevent tumor formation in FGF19-TG mice (23). After 6 months, mice were necropsied and livers were excised for analysis. The livers of mice treated with the control antibody had macroscopically apparent large nodules on multiple lobes (Fig. 5D). However, livers of mice treated with LD1 (Fig. 5D) or 1A6 had no evidence of neoplasia. We also measured liver weight to assess tumor burden, as this parameter was previously shown to correlate strongly with percent tumor volume in the FGF19-TG model (18,23). The weight of livers from mice treated with LD1 or 1A6 (p=0.035 and p=0.052, respectively) was significantly lower than that from mice treated with control antibody (Fig. 5E). The difference in liver weight between mice treated with LD1 and mice treated with 1A6 was not significant (p = 0.439) (Fig. 5E). Together these data clearly demonstrate the in vivo efficacy of LD1 in inhibiting hepatocellular carcinoma in preclinical models.

FGFR4 expression is altered in cancer. We assessed FGFR4 expression in a variety of human normal and cancerous tissues by analyzing the BioExpress database (GeneLogic, Inc., Gaithersburg, MD, USA). FGFR4 expression is highly variable in most types of cancer. Compared with normal tissues, FGFR4 expression was elevated in liver, colorectal, gastric, esophagus, and testicular cancers, but decreased in kidney, lung, lymphoid, and small bowel cancers (Fig. 6A). Using immunohistochemistry (IHC), we localized FGFR4 in a panel of lung, breast, pancreatic, and ovarian adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, thyroid carcinoma, and normal lung, pancreas, and thyroid samples. FGFR4 detection gave membranous and cytoplasmic staining in normal and neoplastic epithelial cells (a representative example is shown in Figure 6B). Higher levels of staining are often found in tumor samples compared to normal tissue. From pancreas (41% of specimens), breast (46%), lung (31%), ovary (41%), colon (90%), liver (33%), and thyroid (11%) (Table 2 and Moderate to significant anti-FGFR4 labeling was evident in tumors from ref. 23).

Table 2: FGFR4 expression in normal and cancer tissues. Prevalence of FGFR4 expression in normal and cancerous tissues as determined by histopathological assessment of FGFR4 immunostaining.

Extensive expression of FGFR4 in human HCC was also previously confirmed by in situ hybridization (23). Because a link between FGFR4 and HCC has long been suggested, we decided to further evaluate FGFR4 expression in 23 primary human liver tumors and 11 normal livers using quantitative real-time polymerase chain reaction (qRT-PCR). The expression of FGFR4 in each sample was normalized to the expression of this receptor in the first normal liver sample (N1). The average level of FGFR4 in liver tumors (1.22±0.05-fold) was moderately elevated compared with normal liver (0.90±0.04-fold), but the difference did not reach statistical significance when the population was taken as a whole (p=0.23) (Fig. 6C). However, FGFR4 expression was significantly higher (more than 2-fold) in a subset of tumors (7/23; 30%). These results suggest that FGFR4 expression is dysregulated in several types of cancer. The elevated expression of FGFR4 in a subset of liver tumors suggests that it may represent an attractive target for the treatment of liver cancer in a diagnostically selected patient population.

In this study, we provide evidence that FGFR4 is involved in hepatocellular carcinoma and that treatment with an FGFR4-inactivating antibody provides an antitumor effect. To assess the involvement of FGFR4 in liver tumorigenesis, we used a genetically engineered mouse model. Exogenous expression of FGF19 was shown to promote hepatocyte proliferation, hepatocyte dysplasia, and HCC development in mice. Furthermore, we and others demonstrated that Klothoβ is required for the liver-specific activity of FGF19 (16,19,24). Because KLB and FGFR4 are most highly expressed in the liver, we hypothesized that dysregulation of the FGFR4 pathway is responsible for liver tumorigenesis mediated by FGF19. To test this hypothesis, we mated FGF19-TG mice with FGFR4-KO mice. Preneoplastic hepatocyte proliferation and hepatoma formation occurred only in FGF19-TG mice with a FGFR4-WT background. Liver tumorigenesis was abolished in FGFR4-KO mice. We further challenged the mice by administering the potent hepatocarcinogen diethylnitrosamine. Treatment with DEN accelerated HCC development in FGF19-TG mice with an FGFR4-WT background, whereas no evidence of hepatic neoplasia was found in FGFR4-KO mice. A clear conclusion is that FGF19-promoted liver tumorigenesis requires FGFR4.

Together these data suggest a link between FGFR4, liver tumorigenesis, and liver cancer progression. Therefore, FGFR4 is a potential therapeutic target, and its inhibition may provide therapeutic benefit in HCC patients. For this purpose, we developed an anti-FGFR4 neutralizing antibody (LD1). LD1 binds FGFR4 and inhibits ligand binding, pathway activation, regulation of gene expression, cell proliferation, and colony formation (in vitro). The site where LD1 binds FGFR4 was mapped by assessing the interaction of LD1 with FGFR4 constructs carrying point mutations at sites that are similar among FGFR4 orthologs but dissimilar in FGFR1-3 orthologs; FGFR4 These amino acid residues in are replaced with amino acid residues present at equivalent positions in FGFR3. LD1 binds wild-type FGFR4 and all mutant FGFR4 constructs except the G165A mutant. Replacement of glycine at position 165 of FGFR4 with alanine almost abolished LD1 binding. The strong specificity of LD1 for FGFR4 combined with the high identity of this region between FGFRs emphasizes the importance of this residue for LD1 binding. Glycine 165 in FGFR4 corresponds to alanine 171 in FGFR1. Interestingly, alanine 171 is the closest residue in the FGFR1 dimer interface (25). Across the axis of the dimer, the side chain of alanine 171 of one receptor makes hydrophobic contacts with alanine 171 of a neighboring receptor. The sequence conservation in this region of FGFR is consistent with the formation of a receptor-receptor interface by this region (25). Thus, binding of LD1 to this equivalent region in FGFR has the potential to disrupt receptor dimerization. Ligand-induced receptor dimerization is critical for FGFR activation (26,27). Therefore, inhibition of FGFR4 dimerization is a potential mechanism of action of LD1. A similar mechanism of action has been described earlier for other therapeutic antibodies (28).

We show that in vivo, LD1 acts on HCC xenograft tumors by inhibiting the regulation of genes downstream of FGFR4 and by blocking tumor growth. Moreover, administration of LD1 inhibited the formation and occurrence of HCC in FGF19-TG mice.

These data demonstrate that FGFR4 is involved in promoting tumorigenesis and cancer progression. In particular, our results suggest that FGFR4 may play an important role in HCC. Several lines of evidence support this hypothesis. FGFR4 is the predominant FGFR isoform present in human hepatocytes (15). We previously reported that liver tissue has the highest levels of FGFR4 and KLB transcripts, both of which are critical for ligand-stimulated activity through this signaling complex (16). Furthermore, ectopic expression of FGF19, a specific ligand for FGFR4, in mice promotes hepatocyte proliferation, hepatocyte dysplasia, and neoplasia (18), and it has been reported that FGF19-induced hepatocyte proliferation is exclusively mediated by FGFR4 of (24). A recent report suggested that FGFR4 also contributes significantly to HCC progression by regulating AFP secretion, proliferation, and anti-apoptosis (17). FGFR4 expression has also been shown to promote resistance to chemotherapy (29). It should be noted that one group reported a protective but not HCC-promoting effect of FGFR4 in mice (30). It is possible that contextual factors, including ligand identity and concentration as well as co-receptor expression and levels of FGFR, may modulate the role of FGFR4 in tumorigenesis. For example, we found that FGFR4 expression was significantly elevated in a subset of primary liver tumors, suggesting that FGFR4 may represent an attractive target for the treatment of liver cancer in a diagnostically selected patient population. Given the growing evidence that FGFR4 is involved in liver tumorigenesis and HCC progression, we believe that therapeutic intervention including anti-FGFR4 neutralizing antibodies may be beneficial in HCC treatment.

partial reference list

1. Ornitz, D.M., and Itoh, N. 2001. Fibroblast growth factors. Genome Biol 2: REVIEWS3005.

2. Eswarakumar, V.P., Lax, I., and Schlessinger, J. 2005. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev16:139-149.

3. Powers, C.J., McLeskey, S.W., and Wellstein, A. 2000. Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7:165-197.

4.Bange, J., Prechtl, D., Cheburkin, Y., Specht, K., Harbeck, N., Schmitt, M., Knyazeva, T., Muller, S., Gartner, S., Sures, I ., et al. 2002. Cancer progression and tumor cell motility are associated with the FGFR4Arg(388) allele. Cancer Res62:840-847.

5. Cappellen, D., De Oliveira, C., Ricol, D., de Medina, S., Bourdin, J., Sastre-Garau, X., Chopin, D., Thiery, J.P., and Radvanyi, F. 1999. Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat Genet 23:18-20.

6. Chesi, M., Brents, L.A., Ely, S.A., Bais, C., Robbiani, D.F., Mesri, E.A., Kuehl, W.M., and Bergsagel, P.L. 2001. Activated fibroblast growth factor receptor3 is an oncogene that contributes to tumor progression in multiple myeloma. Blood 97:729-736.

7. Chesi, M., Nardini, E., Brents, L.A., Schrock, E., Ried, T., Kuehl, W.M., and Bergsagel, P.L. 1997. Frequent translocation t(4;14)(p16.3;q32 .3) in multiple myeloma is associated with increased expression and activating mutations offibroblast growth factor receptor 3. Nat Genet 16:260-264.

8. Gowardhan, B., Douglas, D.A., Mathers, M.E., McKie, A.B., McCracken, S.R., Robson, C.N., and Leung, H.Y. 2005. Evaluation of the fibroblast growth factor system as a potential target for therapy in human prostate cancer. Br J Cancer 92:320-327.

9. Jaakkola, S., Salmikangas, P., Nylund, S., Partanen, J., Armstrong, E., Pyrhonen, S., Lehtovirta, P., and Nevanlinna, H.1993. Amplification of fgfr4gene in human breast and gynecological cancers. Int J Cancer 54:378-382.

10. Jang, J.H., Shin, K.H., and Park, J.G. 2001. Mutations in fibroblast growth factor receptor2 and fibroblast growth factor receptor3 genes associated with human gastric and colorectal cancers. Cancer Res61:3541-3543.

11. Jang, J.H., Shin, K.H., Park, Y.J., Lee, R.J., McKeehan, W.L., and Park, J.G. 2000. Novel transcripts of fibroblast growth factor receptor 3 reveal aberrant splicing and activation of cryptic splice sequences in colorectal cancer. Cancer Res 90 4052.

12. Jeffers, M., LaRochelle, W.J., and Lichenstein, H.S. 2002. Fibroblast growth factors in cancer: therapeutic possibilities. Expert Opin Ther Targets 6: 469-482.

13. Xiao, S., Nalabolu, S.R., Aster, J.C., Ma, J., Abruzzo, L., Jaffe, E.S., Stone, R., Weissman, S.M., Hudson, T.J., and Fletcher, J.A. 1998. FGFR1isfused with a novel zinc-finger gene, ZNF198, in the t(8;13) leukaemia/lymphomasyndrome. Nat Genet 18:84-87.

14. Shariff, M.I., Cox, I.J., Gomaa, A.I., Khan, S.A., Gedroyc, W., and Taylor-Robinson, S.D. 2009. Hepatocellular carcinoma: current trends inworldwide epidemiology, risk factors, diagnosis and therapeutics. Expert Rev 3 pastroenterol 3 -367.

15. Kan, M., Wu, X., Wang, F., and McKeehan, W.L. 1999. Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase. J Biol Chem274:15947-15952.

16. Lin, B.C., Wang, M., Blackmore, C., and Desnoyers, L.R. 2007. Liver-specific activities of FGF19 require Klotho beta. J Biol Chem282:27277-27284.

17. Ho, H.K., Pok, S., Streit, S., Ruhe, J.E., Hart, S., Lim, K.S., Loo, H.L., Aung, M.O., Lim, S.G., and Ullrich, A. 2009. Fibroblast growth factor receptor4regulates proliferation, anti-apoptosis and alpha-fetoprotein secretion during hepatocellular carcinoma progression and represents a potential target for therapeutic intervention. J Hepatol50:118-127.

18. Nicholes, K., Guillet, S., Tomlinson, E., Hillan, K., Wright, B., Frantz, G.D., Pham, T.A., Dillard-Telm, L., Tsai, S.P., Stephan, J.P., et al. 2002. A mousemodel of hepatocellular carcinoma: ectopic expression of fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am J Pathol 160: 2295-2307.

19. Kurosu, H., Choi, M., Ogawa, Y., Dickson, A.S., Goetz, R., Eliseenkova, A.V., Mohammadi, M., Rosenblatt, K.P., Kliewer, S.A., and Kuro-o, M. 2007. Tissue-specific expression of betaKlotho and fibroblast growth factor (FGF) receptor isoforms determines metabolic activity of FGF19and FGF21. JBiol Chem282:26687-26695.

20. Pai, R., Dunlap, D., Qing, J., Mohtashemi, I., Hotzel, K., and French, D.M. 2008. Inhibition of fibroblast growth factor 19 reduces tumor growth by modulating beta-catenin signaling. Cancer Res68:5086 -5095.

21. Ornitz, D.M., Yayon, A., Flanagan, J.G., Svahn, C.M., Levi, E., and Leder, P.1992. Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitochondria in whole cells. Mol Cell Biol 12:240-247.

22. Holt, J.A., Luo, G., Billin, A.N., Bisi, J., McNeill, Y.Y., Kozarsky, K.F., Donahee, M., Wang da, Y., Mansfield, T.A., Kliewer, S.A., et al. 2003. Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis. Genes Dev17:1581-1591.

23. Desnoyers, L.R., Pai, R., Ferrando, R.E., Hotzel, K., Le, T., Ross, J., Carano, R., D'Souza, A., Qing, J., Mohtashemi, I .,et al.2008.Targeting FGF19inhibits tumor growth in colon cancer xenograft and FGF19transgenichepatocellular carcinoma models.Oncogene27:85-97.

24. Wu, X., Ge, H., Gupte, J., Weiszmann, J., Shimamoto, G., Stevens, J., Hawkins, N., Lemon, B., Shen, W., Xu, J. ., et al.2007. Co-receptor requirements for fibroblast growth factor-19 signaling. J Biol Chem282:29069-29072.

25. Plotnikov, A.N., Schlessinger, J., Hubbard, S.R., and Mohammadi, M. 1999. Structural basis for FGF receptor dimerization and activation. Cell98:641-650.

26. Ibrahimi, O.A., Yeh, B.K., Eliseenkova, A.V., Zhang, F., Olsen, S.K., Igarashi, M., Aaronson, S.A., Linhardt, R.J., and Mohammadi, M. 2005. Analysis of mutations in fibroblast growth factor ( FGF) and a pathogenic mutation in FGFreceptor(FGFR)provides direct evidence for the symmetric two-end model for FGFR dimerization.Mol Cell Biol25:671-684.

27. Mohammadi, M., Olsen, S.K., and Ibrahimi, O.A. 2005. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev16:107-137.

28. Agus, D.B., Akita, R.W., Fox, W.D., Lewis, G.D., Higgins, B., Pisacane, P.I., Lofgren, J.A., Tindell, C., Evans, D.P., Maiese, K., et al. 2002. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2:127-137.

29.Roidl,A.,Berger,H.J.,Kumar,S.,Bange,J.,Knyazev,P.,and Ullrich,A.2009.Resistance to chemotherapy is associated with fibroblast growth factor receptor4up-regulation.Clin Cancer Res15:2058 -2066.

30. Huang, X., Yang, C., Jin, C., Luo, Y., Wang, F., and McKeehan, W.L. 2009. Resident hepatocyte fibroblast growth factor receptor 4 limit shepatocarcinogenesis. Mol Carcinog 48:553-562.

31. Winer, J., Jung, C.K., Shackel, I., and Williams, P.M. 1999. Development and validation of real-time quantitative reversetranscriptase-polymerase chain reaction for monitoring gene expression incardiac myocytes in vitro. Anal Biochem270:41-49 .

32. Tomlinson, E., Fu, L., John, L., Hultgren, B., Huang, X., Renz, M., Stephan, J.P., Tsai, S.P., Powell-Braxton, L., French, D ., et al.2002.Transgenicmice expressing human fibroblast growth factor-19display increased metabolic rate and decreased adiposity.Endocrinology143:1741-1747.

33. Yu, C., Wang, F., Kan, M., Jin, C., Jones, R.B., Weinstein, M., Deng, C.X., and McKeehan, W.L. 2000. Elevated cholesterol metabolism and bile acid synthesis in mice lacking Membrane tyrosine kinase receptor FGFR4.J BiolChem275:15482-15489.

B. Highly specific off-target binding identified and eliminated during humanization of antibodies against FGF receptor 4

Materials and methods

FGFR4 was biotinylated using Sulfo-NHS-LC-Biotin (Pierce; catalog 21335).

Generation of chimeric LD1 – Balb/c mice were immunized with human FGF receptor 4 (FGFR4) ectodomain expressed in CHO cells and purified as described in 14 as described herein. LD1 expressing hybridomas were identified when clones were screened for the ability to block FGF19 binding to FGFR4 in a protein-based ELISA.

Mouse LD1 variable domains were cloned from total RNA extracted from LD1 producing hybridoma cells using standard methods. The light chain was amplified using RT-PCR with degenerate reverse primers for the light chain constant domain (CL) and the heavy chain first constant domain (CH1) and forward primers specific for the N-terminal amino acid sequences of the VL and VH regions Variable domain (VL) and heavy chain variable domain (VH). These variable domains were then cloned in frame into vectors containing the corresponding human light and heavy chain constant regions.

Humanization and affinity maturation of LD1 – Grafting of hypervariable regions of LD1 into the human kappa I (huKI) and human VH subgroup III (huIII) variable domain frameworks used in trastuzumab. Framework repair was used to optimize FGFR4 binding affinity by adding mouse vernier positions until the minimal combination of framework changes that fully restored FGFR4 binding affinity was identified ³³ .

hLD1.vB displayed on phage as a monovalent Fab-P3 fusion was affinity matured using a soft randomization strategy. Sequence diversity was introduced into each hypervariable region separately so that the bias toward murine hypervariable region sequences was maintained using a contaminating oligonucleotide synthesis ^strategy34 . For each variegated position, the codon encoding the wild-type amino acid was contaminated with a 70-10-10-10 nucleotide mixture, resulting in an average 50% mutation rate at each position.

The hLD1.vB diverse phage library was panned using a soluble selection method ³⁵ . This approach relies on a short binding period of low concentrations of biotinylated FGFR4 in solution, followed by a short 5 min capture of phage-bound FGFR4 on immobilized neutravidin. An excess of unlabeled FGFR4 (over 100 nM) was added prior to the capture step to increase dissociation selection stringency. Bound phage were eluted by incubating the wells with 100 mM HCl for 30 min, neutralized with 1 M Tris pH 8, and amplified using XL1-Blue cells and M13/KO7 helper phage. Phage antibodies were reformatted into full-length IgG, transiently expressed in mammalian cells, and purified by protein A chromatography.

Affinity Determination - Affinity determination was performed by surface plasmon resonance using a BIAcore ^™ -2000. Approximately 50 RU hLD1.vB IgG was immobilized in 10 mM sodium acetate pH 4.8 on a CM5 sensor chip, and serial 2-fold dilutions (0.48-1000 nM) of FGFR4 in PBST were injected at a flow rate of 30 μl/min. Each sample was analyzed for 4 minutes association and 10 minutes dissociation. After each injection, the chip was regenerated using 10 mM glycine pH 1.7. Binding responses were corrected for by subtracting RUs from flow cells immobilized with irrelevant IgG at similar densities. Kinetic analyzes were performed using a 1:1 Languir model fitting both kon and koff.

Xenograft experiments – All animal protocols were approved by the Genentech Research Animal Care and Use Committee. Seven-week-old female nu/nu (nude-CRL) mice were obtained from Charles River Laboratories International (strain code 088). Mice were maintained under specific pathogen-free conditions. HUH7 cells ( ^5x106 ; Japan Health Science Research Resources Bank, catalog JCRB0403) were implanted subcutaneously in the flank of mice in HBSS/Matrigel (1:1 v/v; BD Biosciences, catalog 354234) in a volume of 0.2 mL. Tumors were measured with calipers twice a week, and tumor volume was calculated using the formula: V= ^0.5xLxW2 , where L and W are the length and width of the tumor, respectively. When the mean tumor volume reached 145 mm, mice ^were randomized (n=15) and injected intraperitoneally with 0.2 mL of vehicle (PBS), 30 mg/kg chLD1, 30 mg/kg hLD1.vB, or 30 mg/kg hLD1.v22 Process once a week. After treatment, tumor volumes were measured as described above. Percent tumor growth inhibition (%TGI) was calculated using the following formula, where C = control vehicle group mean tumor volume at day 21 and T = mean volume at day 21 from each group of mice given test treatments: %TGI = 100x((CT)/C). Data were analyzed and differences in tumor doubling between groups were assessed using the log-rank test with JMP software version 6.0 (SAS Institute; Cary, NC). Data are presented as mean tumor volume ± SEM.

Pharmacokinetic studies in mice - NCR nude mice were supplied by Taconic (catalogue NCRNU). C3 knockout mice ³⁶ were backcrossed with C57BL/6 mice for at least 10 generations. Progeny were crossed to generate C3 knockout mice and wild type controls. In this study, they were referred to as C3ko and C3wt mice, respectively.

Mice weighing 15.5-38.3 g were administered a dose of 1, 5 or 20 mg/kg body weight IV bolus of anti-FGFR4 antibody via the tail vein. At selected time points, up to 28 days post-dose, blood samples (n = 3 mice per time point) were collected via retro-orbital bleeding or cardiac puncture and serum was isolated. Serum samples were stored at -80°C until determination of anti-FGFR4 antibody serum concentrations using ELISA.

PK parameters were assessed using anti-FGFR4 antibody serum concentration-time profiles using WinNonlin Enterprise version 5.2.1 (Pharsight Corp.,). Since one concentration-time profile was determined for each group, one estimate for each PK parameter was obtained and reported, along with the standard error of the fit (SE) for each PK parameter. Modeling was performed using the nominal dose administered to each group.

Radioiodination – Antibodies were radioiodinated using the indirect lodogen addition ^method37 . Radiolabeled proteins were purified using NAP5 ^™ columns (GE Healthcare Life Sciences, catalog 17-0853-01 ) pre-equilibrated in PBS. The specific activities of the molecules used in the in vitro studies were 14.38 μCi/μg for chLD1 and 15.05 μCi/μg for hLD1.vB. The specific activities of the molecules used in the in vivo studies were 12.52 μCi/μg for chLD1 and 9.99 μCi/μg for hLD1.vB. After radioiodination, radioiodinated antibodies were characterized by size exclusion high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and ELISA, were intact and retained Antigen binding comparable to unlabeled antibody.

In vitro incubation - For incubation in serum, antibodies were added to NCR nude mouse, C3ko or C3wt mouse serum ³⁶ , and PBS + 0.5% BSA at a final concentration of 200 μg/ml. Aliquots (100 μl) were prepared and incubated at 37 °C with gentle rotation. Samples were transferred to dry ice at 0, 4, 8, 24, 48, and 96 hours and stored at -70°C until ELISA analysis.

For incubation in plasma, antibodies were added to macaque, human, rat (Bioreclamation LLC, catalogs CYNPLLIHP, HMLLIHP, and RATPLLIHP, respectively), and NCR nude mouse plasma (Taconic, catalog NCRNU-E) and PBS+ 0.5% BSA, final concentration 200μg/ml± ¹²⁵ I-antibody, final concentration 5x10 ⁶ CPM/ml. Aliquots (100 μl) were prepared and incubated at 37 °C with gentle rotation. Transfer samples to dry ice at 0, 24, and 48 hours and store at -70°C until analysis by size exclusion HPLC ( ^125I -antibody + unlabeled antibody sample) or protein G extraction followed by SDS-PAGE (unlabeled antibody sample).

Tissue distribution studies – female NCR nude mice received an IV bolus of ^125I -chLD1 (300 μCi/kg) ± unlabeled chLD1 (20 mg/kg) or ^125I -hLD1.vB (300 μCi/kg) ± unlabeled hLD1.vB (20 mg/kg). Blood was collected at 15 minutes and 2, 5, 24, 72, and 120 hours post-dose and processed for serum. Serum was frozen at -70°C until analysis by size exclusion HPLC and protein G extraction followed by SDS-PAGE separation. Total radioactivity counts were also obtained using the Wallac 1480 Wizard 3"(EC&G Wallac, catalog 1480-011). Liver, lung, kidney, and heart were collected at 2, 72, and 120 hours post-dose and frozen at -70°C until analysis Total radioactivity.All animals were handled according to IACUC guidelines.

IgG and FGFR4 ELISA – FGFR4-specific IgG was measured using the FGFR4 ELISA. To immobilize FGFR4 on a microtiter plate, add in Magic buffer + 0.35M NaCl (1x PBSpH7.4, 0.5%BSA, 0.05%Tween-20, 0.25%Chaps, 5mM EDTA, 0.2%BgG, 0.35M NaCl, 15ppm LD1 standards (chLD1, hLD1.vB, or hLD1.v22) and samples diluted in Proclin) and tested with F(ab') ₂ goat anti-huIgG Fc conjugated to horseradish peroxidase (HRP, Jackson, Prod. catalog 109-036-098) detection capture IgG.

Total antibodies were determined using a human Fc ELISA by incubating diluted samples on microtiter plates coated with F(ab') ₂ rabbit anti-huIgG Fc (Jackson, Cat. F(ab') ₂ goat anti-huIgG Fc for HRP (Jackson, Cat. No. 109-036-098) assay. Color development was performed using TMB peroxidase substrate solution (Moss, catalog number _TMBE -1000), and the reaction was terminated by the addition _of 1M H3PO4. Plates were read at 450/620 nm on a microplate reader (Biotek EL311 or equivalent).

Size Exclusion HPLC – Size Exclusion HPLC was performed in PBS using a Phenomenex ^™ BioSep-SEC-S3000 column (300x7.8 mm, 5 μm column; Torrance, catalog 00H-2146-KO), with samples starting at pH 4.0 and pH 7. 0. Samples at pH 7.0 were diluted in PBS; samples at pH 4.0 were generated by lowering the pH with 200 mM citric acid pH 3.0. The flow rate was 0.5ml/min for 30 minutes, isocratic. The ChemStation analog-to-digital converter was set to 25,000 U/mV, peak width 2 seconds, gap 4 nM (Agilent Technologies; catalog 35900E). Radioactivity was detected with a raytest Ramona90 (raytest USA Inc.; Wilmington, NC) coupled to a standard Agilent 1100HPLC module system (Santa Clara, CA).

Protein G bead extraction and SDS-PAGE separation – Triton X-100 (final concentration 1% (v/v)) was added to fractions collected from size exclusion HPLC, followed by protein G beads (GE Lifesciences Inc, catalog 17-0885-01). Samples were incubated overnight at 4°C with gentle rotation, then beads were washed four times with PBS + 1% Triton X-100. Split each sample and use half of the samples Sample reduction reagent (catalogue NP0004) for reduction. use 4x LDS sample buffer (pH8.4) (Catalogue NP00007) ± Treat beads with sample reducing reagent and incubate at 99°C for 5 minutes; then use 1x MOPS SDS running buffer (catalog NP0001) applied to 4-12% Bis-Tris gel (catalogue NP0321BOX). The gel was stained with Coomassie blue R250 dye. all Reagents were obtained from Invitrogen.

Mass Spectrometry and Bioinformatics Analysis - Samples excised from SDS- ^PAGE were processed as previously described38. Briefly, following fast-solution microwave-assisted trypsin digestion, peptides were separated by reverse-phase chromatography and eluted directly into a nanospray ionization source with a spray voltage of 2 kV and analyzed using an LTQ XL-Orbitrap mass spectrometer (ThermoFisher) . The precursor ions were analyzed in FTMS at 60,000 resolution. MS/MS was implemented in the LTQ, operating the instrument in data-dependent mode where the top 10 most abundant ions were fragmented. Data were searched using the Mascot search algorithm (Matrix Sciences) or by reinterpretation.

For searching Mascot data: search criteria include full MS tolerance 20ppm, MS/MS tolerance 0.5Da (GlyGly on Lys), methionine oxidation, +57Da on Cys and phosphorylation of ST with Y as variable modification , with up to 3 miscuts. Data were searched against the mammalian subset of the Swissprot database.

result

Humanization and standardization of LD1. The human chimeric antibody LD1 (chLD1) has been shown to bind human FGFR4, block signaling by FGF19 and other FGF ligands, and suppress tumor growth in a HUH7 human hepatocellular carcinoma (HCC) xenograft model ¹⁴ . As a first step in LD1 humanization, the light and heavy chain variable domains of chLD1 were compared to the human kappa I (huKI) and human VH subgroup III (huIII) variable domain frameworks used in trastuzumab Yes (Figure 10). Hypervariable regions from chLD1 were grafted into these human variable frameworks to generate direct CDR grafts (hLD1.vA) ^15,16 . The affinity of hLD1.vA was reduced approximately 5-fold when compared to chLD1 binding to FGFR4 by surface plasmon resonance (not shown). Substitution of mouse sequences at multiple fine-tuned positions in both the light and heavy chain variable domains was explored as a means to improve binding and led to the identification of three important mouse fine-tuned positions in the LC: P44F, L46I and Y49S. These changes were introduced in hLD1.vB, which has a comparable affinity for FGFR4 as chLD1 (Table 3).

Table 3: Binding kinetics of anti-FGFR4 antibody variants. On and off rates of human FGFR4-binding immobilized antibody variants were measured using surface plasmon resonance.

Surprisingly, despite similar FGFR4 binding affinities (Fig. 11A), hLD1.vB reduced antitumor efficacy in nu/nu mice in the HUH7 human HCC xenograft model compared to chLD1 (Fig. 11B) . After 9 days, HUH7 tumors from mice treated with PBS had grown to an average volume of approximately 700 mm ³ . In the chLD1-treated group, the mean HUH7 tumor volume was approximately 400 mm ³ , which inhibited tumor growth by 43% compared to tumors in PBS-treated animals. However, the average tumor volume in mice treated with hLD1.vB was approximately 600 mm ³ , which inhibited tumor growth by 14% compared to tumors in PBS-treated animals.

Pharmacokinetic assessment of chLD1 and hLD1.vB in athymic NCR nude mice revealed rapid clearance of both chLD1 and hLD1.vB at 1 mg/kg IV (140 and 132 mL/d/kg, respectively), suggesting that chLD1 and hLD1.vB are cleared by Target-mediated clearance mechanism. This clearance mechanism appears to be saturable for chLD1 at the higher dose of 20 mg/kg. At this dose, the observed clearance (11.7 mL/d/kg; Figure 11C) was in the range of target-independent clearance observed in mice for a typical humanized antibody (6-12 mL/d/kg ) within (ref. 17 and P.Theil personal communication). However, hLD1.vB was still rapidly cleared (34.2 mL/d/kg; Fig. 11C). This suggests that there may be another clearance mechanism for hLD1.vB responsible for the apparent loss of efficacy in the mouse xenograft model.

Consistent with the PK findings, biodistribution studies using125I- ^chLD1 and125I- ^hLD1.vB revealed significantly different distribution profiles (Figure 11D). Due to the high expression of FGFR4 on hepatocytes, ^125I -chLD1 distributed rapidly and specifically to the liver, while only a limited amount ^of125I -hLD1.vB was found in the liver at equivalent doses up to 2 hours (~80 versus 35 %ID/g). In contrast, the distribution observed for these antibodies in blood was reversed, suggesting that there is a competitive interaction preventing distribution of hLD1.vB to the liver as opposed to a loss of antibody stability in vivo that would result in a loss of overall radioactivity.

Identification of C3 interference. Attempting to explain the in vivo differences observed between chLD1 and hLD1.vB, we assessed the stability of the antibodies in plasma and potential off-target plasma or tissue interactions that could affect their function. Plasma stability was assessed by incubating chLD1 or hLD1.vB in mouse, rat, monkey or human plasma for 48 hours at 37°C followed by assessment of both FGFR4 binding activity and total human IgG concentration. While there was no change in total chLD1 or hLD1.vB concentrations measured by IgG ELISA (not shown), hLD1.vB recovery by FGFR4 ELISA was significantly reduced in mouse and rat plasma compared to control incubations in PBS/BSA (decreased ~30%) (Fig. 12A). In comparison, chLD1FGFR4 binding activity was not lost in any of the conditions tested. The marked reduction in hLD1.vB recovery (specifically from rodent plasma) suggests that this loss is not due to degradation, but more likely to the formation of interfering complexes in rodent plasma. Because the interaction of hLD1.vB with mouse plasma can lead to the formation of higher molecular weight complexes, iodinated chLD1 and hLD1.vB were also incubated in plasma and analyzed using size exclusion HPLC. High molecular weight peaks were only detected in mouse plasma samples containing125I- ^hLD1.vB , but ^not125I -chLD1. In addition to the expected antibody peak at 150 kDa, peaks corresponding to about 270 and about 550 kDa were initially detected (Figure 12B), however, by 48 hours, only the 150 and 270 kDa peaks remained; the 550 kDa peak was no longer observed. These higher molecular weight peaks were not detected in macaque and human plasma or in PBS/BSA containing hLD1.vB or in any samples containing chLD1 (Figure 15). Interestingly, the presence of these high molecular weight peaks directly correlated with the antibody recovery data obtained in the FGFR4 ELISA. Moreover, the presence of these peaks decreased when the assay was performed at pH 4.0 (Figure 15), further supporting a hLD1.vB-dependent interaction with mouse serum.

Immunoprecipitation of mouse plasma revealed a protein of approximately 37 kDa that was selectively pulled down with hLD1.vB but not with chLD1 (Fig. 12C). Consistent with the findings from size exclusion HPLC, this 37 kDa protein band was observed in rat, but not macaque and human plasma samples (Fig. 16A-C). Furthermore, this protein was detected in plasma from hLD1.vB-administered mice (Fig. 16D). MS/MS analysis of a tryptic peptide derived from the 37 kDa mouse plasma protein identified this band as derived from mouse complement C3 (Figure 12D). The direct involvement of C3 is supported by complete recovery of hLD1.vB incubated in plasma from C3 knockout (ko) mice (Figure 17).

Reassessment of affinity maturation and C3 binding. Both chLD1 and hLD1.vB share the same human constant regions and complementarity determining regions (CDRs), and thus differ only in their variable domain frameworks. Furthermore, the light and heavy chain variable domain frameworks used in humanized hLD1.vB share a high degree of homology with several humanized antibodies, including Trastuzumab, which has not been reported to exhibit interactions with mouse serum proteins monoclonal antibody. Thus, the off-target interaction of hLD1.vB with mouse C3 most likely arises from the specific combination of mouse LD1 CDRs and human variable domain frameworks.

We reasoned that some changes in the CDR sequences of hLD1.vB (derived from affinity maturation of Fab fragments displayed on phage) could lead to improved affinity for FGFR4 with concomitant loss of mouse C3 binding. Variants were selected as IgG expressing phage and screened for FGFR4 binding affinity and potential interaction with mouse C3 using immunoprecipitation assay coupled to SDS-PAGE analysis. A variant with 3 amino acid changes in CDR-H2 (H52L, S53V and D60E, Figure 10) compared to hLD1.vB, hLD1.v22 showed improved binding affinity for FGFR4 (Table 3) and binding Loss of complement C3b (Fig. 13B) both.

The extent to which mouse complement C3 alters hLD1.vB and hLD1.v22 clearance in vivo was assessed in a pharmacokinetic study comparing C3ko mice to C3wt mice at 20 mg/kg. As previously observed in NCR mice, hLD1.vB was rapidly cleared from circulation (29 mL/d/kg) in C3wt mice; however, both chLD1 and hLD1.vB had similar pharmacokinetics in C3ko mice The clinical profile (clearances were 8.7 and 9.3 mL/d/kg, respectively) (Fig. 14A and Table 4).

Table 4: Pharmacokinetic parameters of anti-FGF.R4 variants dosed IV at 20 mg/kg

These data are consistent with tissue distribution data and confirm that in vivo specific interaction with mouse complement C3 results in rapid clearance of hLD1.vB. Because clearance of hLD1.vB was significantly improved in C3ko mice and hLD1.v22 does not bind C3, we expected that hLD1.v22 would have a similar pharmacokinetic profile to chLD1 in NCR nude mice. As shown in Figure 14B, the clearance of chLD1 and hLD1.v22 in NCR nude mice was similar (11.8 and 11.3 mL/d/kg, respectively), while consistent with our previous findings, hLD1.vB was rapidly cleared ( 46.7mL/d/kg).

The ability of hLD1.v22 to inhibit tumor growth in a HUH7 HCC xenograft model was assessed in comparison with chLD1 and hLD1.vB. After 21 days, HUH7 tumors from mice treated with PBS had grown to an average volume of approximately 2,100 mm ³ ( FIG. 14C ). Of the 15 animals in the PBS-treated group, 3 animals were euthanized before the end of the study due to tumor volume limitations (approximately 2,500 mm ³ ). In the hLD1.vB-treated group, the mean HUH7 tumor volume was approximately 1,200 mm ³ , representing a 43% inhibition of tumor growth compared to tumors in PBS-treated animals. However, the average tumor volume in mice treated with hLD1.v22 was approximately 530 mm ³ . This result was comparable to chLD1 -treated mice, where the average HUH7 tumor volume was approximately 350 mm ³ . This represented a 75% and 83% reduction in tumor size compared to the PBS vehicle treated group for both the hLD1.v22 and chLD1 treated groups, respectively. The tumor doubling time of the groups treated with hLD1.vB (12.2 days), hLD1.v22 (15.8 days) or chLD1 (17.1 days) was significantly longer than that of the PBS-treated group (8.2 days). In addition, the tumor doubling time of the hLD1.v22 or chLD1-treated group was significantly longer than that of the hLD1.vB-treated group. The similar in vivo performance of both hLD1.v22 and chLD1 compared to hLD1.vB strongly suggests that specific off-target interactions with mouse complement C3 lead to increased clearance, resulting in lower hLD1.vB exposure and reduced efficacy.

Thus, the generation of anti-FGFR4 antibody hLD1.v22 has the following steps: 1) generation of human chimeric LD1 (chLD1) comprising LD1 murine VL and VH domains and human IgG1 constant domain; 2) grafting of 6 murine HVRs from chLD1 Incorporation of human VL kappa I and human VH subgroup III variable domain frameworks produced direct HCR grafts to generate antibody hLD1.vA. The binding of antibody hLD1.vA to FGFR4 was about 5 times lower than that of ChLD1; 3) The introduction of light chain mutations P44F, L46I and Y49S produced antibody hLD1.vB. Antibody hLD1.vB binds to human FGFR4 about 2-fold weaker than antibody chLD1, but hLD1.vB has reduced in vivo tumor efficacy compared to chLD1, rapid in vivo clearance, and was found to bind mouse complement protein C3; 4) Implemented Affinity maturation and addition of three changes to HVR H2 (H52L, S53V, D60E) improved binding affinity to FGFR4 and abrogated binding to mouse complement c3 protein. The resulting humanized and affinity matured antibody (antibody hLD1.v22) has comparable in vivo potency, pK and tissue distribution to chLD1. Furthermore, it has been determined that the antibody hLD1.v22 has at least comparable biological activity as the parental chLD1 antibody, eg inhibition of cancer in human xenograft tumor studies.

partial reference list

1. James LC, Roversi P, Tawfik DS. Antibody multispecificity mediated by conformational diversity. Science 2003;299:1362-7.

2. Krishnan L, Sahni G, Kaur KJ, Salunke DM. Role of antibody paratope conformational flexibility in the manifestation of molecular mimicry. Biophys J2008;94:1367-76.

3. Notkins AL. Polyreactivity of antibody molecules. Trends Immunol 2004; 25:174-9.

4. Thorpe IF, Brooks CL, 3rd. Molecular evolution of affinity and flexibility in the immune system. Proc Natl Acad Sci U S A2007;104:8821-6.

5. Yin J, Beuscher AEt, Andryski SE, Stevens RC, Schultz PG. Structural plasticity and the evolution of antibody affinity and specificity. J Mol Biol2003;330:651-6.

6. Thielges MC, Zimmermann J, Yu W, Oda M, Romesberg FE. Exploring the energy landscape of antibody-antigen complexes: protein dynamics, flexibility, and molecular recognition. Biochemistry 2008; 47: 7237-47.

7. Feyen O, Lueking A, Kowald A, Stephan C, Meyer HE, Gobel U, et al. Off-target activity of TNF-α inhibitors characterized by protein biochips. Analytical and Bioanalytical Chemistry 2008; 391: 1713-20.

8. Kijanka G, Ipcho S, Baars S, Chen H, Hadley K, Beveridge A, et al. Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays. J Immunol Methods 2009;340:132-7.

9. Lueking A, Beator J, Patz E, Mullner S, Mehes G, Amersdorfer P. Determination and validation of off-target activities of anti-CD44variant6 antibodies using protein biochips and tissue microarrays. Biotechniques2008;45:Pi-v.

10. Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. JMol Biol2007; 368:652-65.

11. Rosenwald S, Kafri R, Lancet D. Test of a statistical model formolecular recognition in biological repertoires. J Theor Biol 2002;216:327-36.

12. James LC, Tawfik DS. The specificity of cross-reactivity: promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness. Protein Sci2003;12:2183-93.

13. Kramer A, Keitel T, Winkler K, Stocklein W, Hohne W, Schneider-Mergener J. Molecular basis for the binding promiscuity of an anti-p24(HIV-1) monoclonal antibody. Cell 1997;91:799-809.

14. French DM, Lin BC, Wang M, K, Bolon B, Ferrando R, et al. Targeting FGFR4 Inhibits Hepatocellular Carcinoma in Preclinical Animal Models. J Clin Invest2011; Submitted.

15. Dennis MS. Humanization by CDR Repair In: Shire S, Gombotz W, Bechtold-Peters K, Andya J, eds. Pharmaceutical Aspects of Monoclonal Antibodies. New York: Co-published by the Association for Pharmaceutical Scientists & Springer, 2010: 9-28.

16. MacCallum RM, Martin ACR, Thornton JT. Antibody-antigen interactions: Contact analysis and binding site topography. Journal of Molecular Biology 1996; 262: 732-45.

17. Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, et al. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 2006;55:717-27.

18. Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies. Mol Cells 2005; 20:17-29.

19. Lutz HU, Jelezarova E. Complement amplification revisited. Mol Immunol 2006; 43:2-12.

20. Sahu A, Lambris JD. Structure and biology of complement protein C3, a connecting link between innate and acquired immunity. Immunological Reviews 2001; 180: 35-48.

21.Manderson AP, Pickering MC, Botto M, Walport MJ, Parish CR.Continual low-level activation of the classical complement pathway. Journal of Experimental Medince2001;194:747-56.

22. Thurman JM, Holers VM. The central role of the alternative complement pathway in human disease. The Journal of Immunology 2006;176:1305-10.

23. Sahu A, Pangburn MK. Covalent attachment of human complement C3to IgG. The Journal of Biological Chemistry 1994; 269: 28997-9002.

24. Vidarte L, Pastor C, Mas S, Blazquez AB, de los Rios V, Guerrero R, et al. Serine132 is the C3 covalent attachment point on the CH1 domain of human IgG1. J Biol Chem2001;276:38217-23.

25. Osmers I, Szalai AJ, Tenner AJ, Barnum SR. Complement in BuB/BnJmice revisited: serum C3 levels and complement opsonic activity are not elevated. Mol Immunol2006;43:1722-5.

26. Otte L, Knaute T, Schneider-Mergener J, Kramer A. Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide3monoclonal antibody. J Mol Recognit2006;19:49-59.

27. Arenkov P, Kukhtin A, Gemmell A, Voloshchuck S, Chupeeva V, Mirzabekov A. Protein microchips: use for immunoassay and enzymatic reactions. Analytical Biochemistry 2000; 278: 123-31.

28. Ge H. UPA, a universal protein array system for quantitative detected protein-protein, protein-DNA, protein-RNA, and protein-ligand interactions. Nucleic Acids Research2000;28:i-vii.

29. Fodor SP, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 1991;251:767-73.

30. Forster R, Valin M, Lemarchand T, Palate B, Fifre A. Validation of tissue cross reactivity (TCR) studies: Tissue cross reactivity of anti-human CD209 in cynomolgus tissues. Toxicology Letters 2009; 189: S100-S.

31. Kallioniemi O-P, Wagner U, Kononen J, Sauter G. Tissue microarray technology for high-throughput molecular profiling of cancer. Human Molecular Genetics 2001; 10:657-62.

32. Lynch C, Grewel I. Preclinical safety evaluation of monoclonal antibodies. In: Chernajovsky Y, Nissim A, eds. Therapeutic Antibodies: Handbook of Experimental Pharmacology. Berlin: Springer-Verlag, 2008: 19-44.

33. Dennis MS. CDR Repair: A novel approach to antibody humaization. In: Shire SJ, Gombotz W, Bechtold-Peters K, Adndya J, eds. Current Trends in Monoclonal Antibody Development and Manufacturing. New York: Springer, 2010: 9- 28.

34. Gallop MA, Barrett RW, Dower WJ, Fodor SPA, Gordon EM. Applications of combinatorial technologies to drug discovery. 1. Background and peptide combinatorial libraries. J Med Chem1994;37:1233-51.

35. Lee CV, Liang W-C, Dennis MS, Eigenbrot C, Sidhu SS, Fuh G. High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Bio2004;340:1073-93.

36. Circolo A, Garnier G, Fukuda W, Wang X, Hidvegi T, Szalai AJ, et al. Genetic disruption of the murine complement C3 promoter region generates deficient mice with extrahepatic expression of C3 mRNA. Immunopharmacology 1999;42:135-49.

37. Chizzonite R, Truitt T, Podlaski FJ, Wolitzky AG, Quinn PM, Nunes P, et al. IL-12:Monocloncal antibodies specific for the 40-kDa subunit blockreceptor binding and biological activity on activated human lymphoblasts. The Journal of Immunology 1991; :1548-56.

38. Jin Z, Li Y, Pitti R, Lawrence D, Pham VC, Lill JR, et al. Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8mediate extrinsicapoptosis signaling. Cell2009;137:721-35.

While the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

Claims (20)

1. combining an antibody for the separation of FGFR4, wherein this antibody comprises: (a) SEQ ID NO:1's HVR-H1 sequence；The HVR-H2 sequence of (b) SEQ ID NO:2；(c) SEQ ID NO:3's HVR-H3 sequence；The HVR-L1 sequence of (d) SEQ ID NO:4；(e) SEQ ID NO:5's HVR-L2 sequence；(f) the HVR-L3 sequence of SEQ ID NO:6.

2. the antibody of claim 1, it comprises the light chain variable of SEQ ID NO:9,10,11 and 12 further Territory Frame sequence, and/or the heavy chain variable domain Frame sequence of SEQ ID NO:13,14,15 and 16.

3. the antibody of claim 1, it comprises VH sequence SEQ ID NO:7 and/or VL sequence SEQ ID NO:8。

4. the antibody of claim 2, it comprises VH sequence SEQ ID NO:7 and/or VL sequence SEQ ID NO:8。

5. the antibody of any one of claim 1-4, it is monoclonal antibody.

6. the antibody of any one of claim 1-4, its behaviour antibody, humanized antibody or chimeric antibody.

7. the antibody of any one of claim 1-4, it is monoclonal antibody, and its behaviour antibody, humanization Antibody or chimeric antibody.

8. the antibody of any one of claim 1-4, it is total length IgG1 antibody.

9. the antibody of claim 5, it is total length IgG1 antibody.

10. the antibody of claim 6, it is total length IgG1 antibody.

The antibody of 11. claim 7, it is total length IgG1 antibody.

12. the nucleic acid separated, the antibody of its coding any one of claim 1-11.

13. 1 kinds of host cells, it comprises the nucleic acid of claim 12.

14. the method producing antibody, it host cell including cultivating claim 13 so that antibody is raw Become.

15. the method for claim 14, it farther includes to reclaim antibody from host cell.

16. 1 kinds of immune conjugates, its antibody comprising any one of claim 1-11 and cytotoxic agent.

17. 1 kinds of pharmaceutical compositions, its antibody comprising any one of claim 1-11 and pharmaceutical acceptable carrier.

The pharmaceutical composition of 18. claim 17, it comprises other therapeutic agent further.

The antibody of 19. any one of claim 1-11 is used for the purposes treating in the medicine of cancer in preparation.

The antibody of 20. any one of claim 1-11 is used for the use suppressed in the medicine of tumor cell proliferation in preparation On the way.